Polypeptides

ABSTRACT

There is provided inter alia a polypeptide comprising an immunoglobulin chain variable domain which binds to TNF-alpha, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1-CDR3 and FR1-FR4 are as defined in the specification.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.15/273,353 filed on Sep. 22, 2016, which is a continuation ofinternational application PCT/EP2016/057021 filed on Mar. 31, 2016, andwhich derives priority from EP 15162112.5 filed on Mar. 31, 2015, thecontents of each of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to polypeptides comprising animmunoglobulin chain variable domain (or ‘variable domain’) which bindsto Tumour Necrosis Factor-alpha (‘TNF-alpha’, ‘TNF-α’ or ‘TNF’) as wellas to constructs and pharmaceutical compositions comprising thesepolypeptides. The present invention also relates to nucleic acidsencoding such polypeptides, to methods for preparing such polypeptides,to cDNA and vectors comprising nucleic acids encoding such polypeptides,to host cells expressing or capable of expressing such polypeptides andto uses of such polypeptides, pharmaceutical compositions or constructs.

BACKGROUND OF THE INVENTION

Tumour necrosis factor-alpha is a homotrimeric pro-inflammatory cytokineinvolved in systemic inflammation which exists in both soluble andmembrane-bound forms. TNF-alpha is secreted predominantly by monocytesand macrophages but is also secreted by tumour cell lines as well asCD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and Bcell lines. TNF-alpha has been implicated in inflammatory diseases,autoimmune diseases, viral, bacterial and parasitic infections,malignancies, and/or neurodegenerative diseases and is a target forspecific biological therapy in autoimmune/autoinflammatory diseases suchas rheumatoid arthritis and Crohn's disease.

Crohn's disease, also known as Crohn syndrome and regional enteritis, isa type of inflammatory bowel disease causing a wide variety of symptoms.It primarily causes abdominal pain, diarrhea, vomiting and/or weightloss but may also cause complications outside the gastrointestinal tract(GIT) such as anaemia, skin rashes, arthritis, inflammation of the eye,tiredness, and lack of concentration (Baumgart et al 2012 The Lancet380(9853):1590-605, herein incorporated by reference in its entirety).Crohn's disease is a presently incurable life-long gastrointestinaldisease that is difficult to control with conventional therapies.Crohn's disease is discussed in more detail below under ‘autoimmunediseases’.

A TNF-alpha inhibitor which has sufficient specificity to TNF-alpha maybe an efficient prophylactic or therapeutic pharmaceutical forpreventing or treating diseases such as Crohn's disease, where TNF-alphahas been implicated as a key cytokine driving the pathology observed.

Antibody-based therapeutics have significant potential as effectivetreatments for autoimmune disease because they have high specificity fortheir target and a low inherent toxicity. Methods of treating autoimmunedisease by administration of an antibody which binds TN F-alpha havebeen described (Kamm et al 2011 Inflamm Bowel Dis 17:2366-91, hereinincorporated by reference in its entirety).

Three anti-TN F-alpha antibodies infliximab (trade name Remicade),adalimumab (trade name Humira) and certolizumab (or ‘certolizumabpegol’, both trade name Cimzia) are used clinically for the treatment ofCrohn's disease; however these antibodies are generally considered to beunsuitable for administration as oral therapeutics due to their inherentinstability and susceptibility to proteolytic degradation by thedigestive system, inflammatory proteases present at the sites ofpathology in the intestinal tract, and intestinal microflora. Theseagents therefore have to be administered by intravenous infusion orsubcutaneous injection which requires specialist training in order touse a hypodermic syringe or needle correctly and safely. These agentsalso require sterile equipment, a liquid formulation of the therapeuticpolypeptide, vial packing of said polypeptide in a sterile and stableform and a suitable site on the subject for entry of the needle.Subjects commonly experience psychological stress before receiving aninjection and pain while receiving an injection. Long term treatmentwith these systemic anti-TN F-alpha antibodies carries increased risksof serious infection and cancer. Together with the high costs ofproduction, these factors currently restrict use of these agents topatients with more severe disease.

Several small molecule anti-inflammatory and immunosuppressive drugs arealso currently in clinical development for Crohn's disease (Danese 2012Gut 61:918-932 and Shealy et al 2010 mAbs 2:428-439, herein incorporatedby reference in its entirety). Although these drugs are orallyadministered, many will be absorbed systemically after administrationand may therefore have systemic immunosuppressive actions that areunrelated to actions against the gastrointestinal tract lesions.Furthermore, as small molecules lack the specificity of antibodies therisk of significant off target side-effects remains high.

Crohn's disease is primarily a disease of the gastrointestinal tract.The production of TNF-alpha is localised to cells present within mucosaland sub-mucosal tissues and this drives chronic inflammatory processeswithin the gut wall and the recruitment of additional inflammatory cellsthat are responsible for development of the disease immunopathology (vanDeventer 1999 Ann Rheum Dis 58(Suppl I):1114-1120). The ability todeliver an oral therapeutic agent with high selectivity for TNF-alpha,but with exposure and activity limited to the gut, may offer efficacysimilar to injectable anti-TNF-alpha antibodies, combined withsignificant improvements in safety due to reduced systemic exposure.

WO 2004/041862, WO 2006/122786 and Coppieters et al 2006 Arthritis &Rheumatism, 54(6):1856-1866 (herein incorporated by reference in theirentirety) disclose single domain antibodies directed against TNF-alphaand related aspects. The sequence referred to in WO 2006/122786 as“TNF1”, “PMP1C2” or “SEQ ID NO: 52”) is characterised further below.

Polypeptides of the present invention may, in at least some embodiments,have one or more of the following advantages compared to anti-TNF-alphasubstances of the prior art:

-   -   (i) increased affinity for TNF-alpha;    -   (ii) increased specificity for TNF-alpha;    -   (iii) increased neutralising capability against TNF-alpha;    -   (iv) increased cross-reactivity with TNF-alpha from different        species such as human and cynomolgus monkey;    -   (v) increased cross-reactivity with both soluble and membrane        forms of TNF-alpha;    -   (vi) reduced immunogenicity, for example when administered to a        mouse, cynomolgus monkey or human;    -   (vii) increased stability in the presence of proteases, for        example (a) in the presence of proteases found in the small        and/or large intestine and/or IBD inflammatory proteases, for        example trypsin, chymotrypsin, MMP3, MMP10, MMP12, other MMPs        and cathepsin and/or (b) in the presence of proteases from gut        commensal microflora and/or pathogenic bacteria, actively        secreted and/or released by lysis of microbial cells found in        the small and/or large intestine;    -   (viii) increased stability to protease degradation during        production (for example resistance to yeast proteases)    -   (ix) increased suitability for oral administration;    -   (x) increased suitability for local delivery to the intestinal        tract and lamina propria following oral administration;    -   (xi) increased suitability for expression, in a heterologous        host such as bacteria such as Escherichia coli, or a yeast        belonging to the genera Aspergillus, Saccharomyces,        Kluyveromyces, Hansenula or Pichia, such as Saccharomyces        cerevisiae or Pichia pastoris;    -   (xii) suitability for, and improved properties for, use in a        pharmaceutical;    -   (xiii) suitability for, and improved properties for, use in a        functional food;    -   (xiv) improved tissue penetration such as penetration of        inflamed colonic mucosal epithelium and submucosal tissues to        access the sub mucosal lamina propria;    -   (xv) remain substantially active after (a) freezing and thawing        and/or (b) after long term storage in lyophilised, liquid/cream        format at for example 37 or 50 degrees C.;    -   (xvi) decreased immunogenicity in humans for example due to        increased sequence similarity to human immunoglobulins;    -   (xvii) increased suitability for formatting in a multispecific        format;    -   (xviii) binding to novel epitopes.

Advantages (i) to (xviii) above may potentially be realised by thepolypeptides of the present invention in a monovalent format or in amultivalent format such as a bihead format (for example homobihead orheterobihead formats).

SUMMARY OF THE INVENTION

The present inventors have produced surprisingly advantageouspolypeptides comprising immunoglobulin chain variable domains which bindto TNF-alpha. These polypeptides in particular benefit from surprisinglyhigh potency. They also neutralise both the soluble and membrane formsof TNF-alpha, are capable of cross-reacting with cynomolgus monkeyTNF-alpha and remain stable on exposure to trypsin, chymotrypsin and/orproteases of the small and large intestine. In one embodiment, thesepolypeptides have undergone further enhancement by engineering. Thesefurther enhanced polypeptides benefit from the above advantages, retaintheir TNF-alpha-neutralising activity during passage through theintestinal tract and further resist degradation and/or inactivation byproteases of the intestinal tract, for example, digestive, inflammatoryand microbial proteases from, for example, multiple mammalian species(rodent, pig, non-human primate and human).

It may be expected that these polypeptides have particular utility inthe prevention or treatment of autoimmune and or inflammatory diseasesuch as inflammatory bowel disease (for example Crohn's disease orulcerative colitis), or in the prevention or treatment of mucositis,particularly when administered orally.

The present invention provides a polypeptide comprising animmunoglobulin chain variable domain which binds to TNF-alpha, whereinthe immunoglobulin chain variable domain comprises three complementaritydetermining regions (CDR1-CDR3) and four framework regions (FR1-FR4),wherein CDR1 comprises a sequence sharing 60% or greater sequenceidentity with SEQ ID NO: 1, CDR2 comprises a sequence sharing 50% orgreater sequence identity with SEQ ID NO: 2 and (a) CDR3 comprises asequence sharing 80% or greater sequence identity with SEQ ID NO: 3 or(b) CDR3 comprises a sequence sharing 50% or greater sequence identitywith SEQ ID NO: 3 and wherein the residue of CDR3 corresponding toresidue number 3 of SEQ ID NO: 3 is R, D, N, C, E, Q, G, H, I, L, K, M,F, P, S, T, W, Y or V.

Also provided is a polypeptide comprising an immunoglobulin chainvariable domain which binds to TNF-alpha, wherein the immunoglobulinchain variable domain comprises three complementarity determiningregions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1comprises a sequence sharing 60% or greater sequence identity with SEQID NO: 15 CDR2 comprises a sequence sharing 50% or greater sequenceidentity with SEQ ID NO: 16 and CDR3 comprises a sequence sharing 50% orgreater sequence identity with SEQ ID NO: 17.

Also provided is a polypeptide comprising an immunoglobulin chainvariable domain which binds to TNF-alpha, wherein the immunoglobulinchain variable domain comprises three complementarity determiningregions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3comprises a sequence sharing 80% or greater sequence identity with SEQID NO: 3.

DESCRIPTION OF THE FIGURES

FIG. 1 —% TNF-alpha neutralising activity of periplasmic supernatantsand purified samples when exposed and not exposed to trypsin andchymotrypsin

FIG. 2A—hs-TNF-alpha neutralization by Q65B1, Q65C7, Q62E10 andadalimumab in HEK-Nf-kB-SEAP reporter assay

FIG. 2B—hs-TNF-alpha neutralization by Q65B1 and Q65D3

FIG. 3—Immunoglobulin chain variable domains in mouse small intestinaland human faecal digests

FIG. 4A—hs-TNF-alpha neutralisation by ID32F, ID34F, Q65B1 andinfliximab (first experiment)

FIG. 4B—hs-TNF-alpha neutralisation by ID32F, ID34F and Q65B1 (secondexperiment)

FIG. 4C—hs-TNF-alpha neutralization by ID37F, ID38F and Remicade (secondexperiment)

FIG. 5A—Stability of ID34F and ID25F in IBD proteases

FIG. 5B—Stability of etanercept and adalimumab in IBD proteases

FIG. 5C—Stability of infliximab in IBD proteases

FIG. 6—Neutralisation of h-TNF-alpha by anti-TNF-alpha polypeptides ofthe prior art

FIG. 7—Calculated luminal [anti-TN F ICVD] in cynomolgus monkeygastrointestinal tract sections

FIG. 8—Total % recovery of anti-TNF ICVD from cynomolgus monkeygastrointestinal tracts

FIG. 9—Humira competition ELISA OD450 data

FIG. 10—anti-TNF ICVD concentration in pooled cynomolgus monkey faeces

FIG. 11—Calculated anti-TN F ICVD recovered from pooled cynomolgusmonkey faeces

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—Polypeptide sequence of ID38F CDR1

SEQ ID NO: 2—Polypeptide sequence of ID38F CDR2

SEQ ID NO: 3—Polypeptide sequence of ID38F CDR3

SEQ ID NO: 4—Polypeptide sequence of ID38F FR1

SEQ ID NO: 5—Polypeptide sequence of ID38F FR2

SEQ ID NO: 6—Polypeptide sequence of ID38F FR3

SEQ ID NO: 7—Polypeptide sequence of ID38F FR4

SEQ ID NO: 8—Polypeptide sequence of ID38F

SEQ ID NO: 9—Polypeptide sequence of soluble human TNF-alpha (monomer)

SEQ ID NO: 10—Polypeptide sequence of membrane-bound human TNF-alpha(monomer)

SEQ ID NO: 11—Polypeptide sequence of soluble cynomolgus monkeyTNF-alpha (monomer)

SEQ ID NO: 12—Polypeptide sequence of membrane-bound cynomolgus monkeyTNF-alpha (monomer)

SEQ ID NO: 13—Polypeptide sequence of soluble mouse TNF-alpha (monomer)

SEQ ID NO: 14—Polypeptide sequence of membrane-bound mouse TNF-alpha(monomer)

SEQ ID NO: 15—Polypeptide sequence of Q62E10 CDR1

SEQ ID NO: 16—Polypeptide sequence of Q62E10 CDR2

SEQ ID NO: 17—Polypeptide sequence of Q62E10 CDR3

SEQ ID NO: 18—Polypeptide sequence of Q62E10 FR1

SEQ ID NO: 19—Polypeptide sequence of Q62E10 FR2

SEQ ID NO: 20—Polypeptide sequence of Q62E10 FR3

SEQ ID NO: 21—Polypeptide sequence of Q62E10 FR4

SEQ ID NO: 22—Polypeptide sequence of Q62E10

SEQ ID NO: 23—Polypeptide sequence of Q65F2

SEQ ID NO: 24—Polypeptide sequence of Q65F3

SEQ ID NO: 25—Polypeptide sequence of Q62F2

SEQ ID NO: 26—Polypeptide sequence of Q65G1

SEQ ID NO: 27—Polypeptide sequence of Q65H6

SEQ ID NO: 28—Polypeptide sequence of Q65F1

SEQ ID NO: 29—Polypeptide sequence of Q65D1

SEQ ID NO: 30—Polypeptide sequence of Q65C7

SEQ ID NO: 31—Polypeptide sequence of Q65D3

SEQ ID NO: 32—Polypeptide sequence of Q65B1

SEQ ID NO: 33—Polypeptide sequence of Q65F6

SEQ ID NO: 34—Polypeptide sequence of Q65F11

SEQ ID NO: 35—Polypeptide sequence of Q65E12

SEQ ID NO: 36—Polypeptide sequence of Q65C12

SEQ ID NO: 37—Polypeptide sequence of Q65A6

SEQ ID NO: 38—Polypeptide sequence of Q65A3

SEQ ID NO: 39—Polypeptide sequence of Q62F10

SEQ ID NO: 40—Polypeptide sequence of Q62F11

SEQ ID NO: 41—Polypeptide sequence of ID7F-EV

SEQ ID NO: 42—Polypeptide sequence of ID8F-EV

SEQ ID NO: 43—Polypeptide sequence of ID9F-EV

SEQ ID NO: 44—Polypeptide sequence of ID13F-EV

SEQ ID NO: 45—Polypeptide sequence of ID14F-EV

SEQ ID NO: 46—Polypeptide sequence of ID15F-EV

SEQ ID NO: 47—Polypeptide sequence of ID22F

SEQ ID NO: 48—Polypeptide sequence of ID23F

SEQ ID NO: 49—Polypeptide sequence of ID24F

SEQ ID NO: 50—Polypeptide sequence of ID25F

SEQ ID NO: 51—Polypeptide sequence of ID26F

SEQ ID NO: 52—Polypeptide sequence of ID27F

SEQ ID NO: 53—Polypeptide sequence of ID28F

SEQ ID NO: 54—Polypeptide sequence of ID29F

SEQ ID NO: 55—Polypeptide sequence of Q62E10-DVQLV

SEQ ID NO: 56—Polypeptide sequence of ID34F

SEQ ID NO: 57—Polypeptide sequence of ID37F

SEQ ID NO: 58—Polynucleotide sequence of 3′ Primer with Spe site

SEQ ID NO: 59—Polypeptide sequence of Q65F1 CDR1

SEQ ID NO: 60—Polypeptide sequence of Q65D1 CDR1

SEQ ID NO: 61—Polypeptide sequence of ID27F CDR2

SEQ ID NO: 62—Polypeptide sequence of ID28F CDR2

SEQ ID NO: 63—Polypeptide sequence of Q65F2 CDR2

SEQ ID NO: 64—Polypeptide sequence of Q65F3 CDR2

SEQ ID NO: 65—Polypeptide sequence of Q62F2 CDR2

SEQ ID NO: 66—Polypeptide sequence of Q65F1 CDR2

SEQ ID NO: 67—Polypeptide sequence of Q65D1 CDR2

SEQ ID NO: 68—Polypeptide sequence of Q65D3 CDR2

SEQ ID NO: 69—Polypeptide sequence of Q65B1 CDR2

SEQ ID NO: 70—Polypeptide sequence of Q65F2 CDR3

SEQ ID NO: 71—Polypeptide sequence of Q65F1 CDR3

SEQ ID NO: 72—Polypeptide sequence of Q65D3 CDR3

SEQ ID NO: 73—Polypeptide sequence of Q65F6 CDR2

SEQ ID NO: 74—Polypeptide sequence of Q65F11 CDR2

SEQ ID NO: 75—Polypeptide sequence of Q65C12 CDR2

SEQ ID NO: 76—Polypeptide sequence of Q65A6 CDR2

SEQ ID NO: 77—Polypeptide sequence of Q65A3 CDR2

SEQ ID NO: 78—Polypeptide sequence of Q65F6 CDR3

SEQ ID NO: 79—Polypeptide sequence of Q65F11 CDR3

SEQ ID NO: 80—Polypeptide sequence of Q62F10 CDR3

SEQ ID NO: 81—Polynucleotide sequence of M13.rev

SEQ ID NO: 82—Polynucleotide sequence of M13.fw

SEQ ID NO: 83—Polynucleotide coding sequence of ID38F, codon optimisedfor yeast expression

SEQ ID NO: 84—Polynucleotide coding sequence of Q62E10, codon optimisedfor yeast expression

SEQ ID NO: 85—Polynucleotide coding sequence of ID38F, codon optimisedfor E. coli 25 expression

SEQ ID NO: 86—Polynucleotide coding sequence of Q62E10, codon optimisedfor E. coli expression

SEQ ID NO: 87—Polynucleotide consisting of ID38F open reading frame forE. coli expression (PelB leader to c-myc-6His tag 2× stop codon)

SEQ ID NO: 88—Polynucleotide consisting of ID38F open reading frame forE. coli expression (PelB leader to Flag-6His tag 2× stop codon)

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Including Antibodies and Antibody Fragments Including theVH and VHH

A conventional antibody or immunoglobulin (Ig) is a protein comprisingfour polypeptide chains: two heavy (H) chains and two light (L) chains.Each chain is divided into a constant region and a variable domain. Theheavy chain variable domains are abbreviated herein as VHC, and thelight (L) chain variable domains are abbreviated herein as VLC. Thesedomains, domains related thereto and domains derived therefrom, arereferred to herein as immunoglobulin chain variable domains. The VHC andVLC domains can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDRs”), interspersed withregions that are more conserved, termed “framework regions” (“FRs”). Theframework and complementarity determining regions have been preciselydefined (Kabat et al 1991 Sequences of Proteins of ImmunologicalInterest, Fifth Edition U.S. Department of Health and Human Services,NIH Publication Number 91-3242, herein incorporated by reference in itsentirety). In a conventional antibody, each VHC and VLC is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The conventional antibody tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains is formed withthe heavy and the light immunoglobulin chains inter-connected by e.g.disulfide bonds, and the heavy chains similarity connected. The heavychain constant region includes three domains, CH1, CH2 and CH3. Thelight chain constant region is comprised of one domain, CL. The variabledomain of the heavy chains and the variable domain of the light chainsare binding domains that interact with an antigen. The constant regionsof the antibodies typically mediate the binding of the antibody to hosttissues or factors, including various cells of the immune system (e.g.effector cells) and the first component (Clq) of the classicalcomplement system. The term antibody includes immunoglobulins of typesIgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the lightchains of the immunoglobulin may be kappa or lambda types. The overallstructure of immunoglobulin-gamma (IgG) antibodies assembled from twoidentical heavy (H)-chain and two identical light (L)-chain polypeptidesis well established and highly conserved in mammals (Padlan 1994 MolImmuno/31:169-217).

An exception to conventional antibody structure is found in sera ofCamelidae. In addition to conventional antibodies, these sera possessspecial IgG antibodies. These IgG antibodies, known as heavy-chainantibodies (HCAbs), are devoid of the L chain polypeptide and lack thefirst constant domain (CH1). At its N-terminal region, the H chain ofthe homodimeric protein contains a dedicated immunoglobulin chainvariable domain, referred to as the VHH, which serves to associate withits cognate antigen (Muyldermans 2013 Annu Rev Biochem 82:775-797,Hamers-Casterman et al 1993 Nature 363(6428):446-448, Muyldermans et al1994 Protein Eng 7(9):1129-1135, herein incorporated by reference intheir entirety).

An antigen-binding fragment (or “‘antibody fragment” or “immunoglobulinfragment”) as used herein refers to a portion of an antibody thatspecifically binds to TNF-alpha (e.g. a molecule in which one or moreimmunoglobulin chains is not full length, but which specifically bindsto TNF-alpha). Examples of binding fragments encompassed within the termantigen-binding fragment include:

(i) a Fab fragment (a monovalent fragment consisting of the VLC, VHC, CLand CH1 domains);

(ii) a F(ab′)2 fragment (a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region);

(iii) a Fd fragment (consisting of the VHC and CH1 domains);

(iv) a Fv fragment (consisting of the VLC and VHC domains of a singlearm of an antibody);

(v) an scFv fragment (consisting of VLC and VHC domains joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VLC and VHC regions pair to formmonovalent molecules);

(vi) a VH (an immunoglobulin chain variable domain consisting of a VHCdomain (Ward et al Nature 1989 341:544-546);

(vii) a VL (an immunoglobulin chain variable domain consisting of a VLCdomain);

(viii) a V-NAR (an immunoglobulin chain variable domain consisting of aVHC domain from chondrichthyes IgNAR (Roux et al 1998 Proc Natl Acad SciUSA 95:11804-11809 and Griffiths et al 2013 Antibodies 2:66-81, hereinincorporated by reference in their entirety)

(ix) a VHH.

The total number of amino acid residues in a VHH or VH may be in theregion of 110-130, is suitably 112-120, and is most suitably 115.

Immunoglobulin chain variable domains of the invention may for examplebe obtained by preparing a nucleic acid encoding an immunoglobulin chainvariable domain using techniques for nucleic acid synthesis, followed byexpression of the nucleic acid thus obtained According to a specificembodiment, an immunoglobulin chain variable domain of the inventiondoes not have an amino acid sequence which is exactly the same as (i.e.shares 100% sequence identity with) the amino acid sequence of anaturally occurring polypeptide such as a VH or VHH domain of anaturally occurring antibody.

The examples provided herein relate to immunoglobulin chain variabledomains per se which bind to TNF-alpha. The principles of the inventiondisclosed herein are, however, equally applicable to any polypeptidecomprising an immunoglobulin chain variable domain which binds toTNF-alpha, such as antibodies and antibody fragments. For example, theanti-TNF-alpha immunoglobulin chain variable domains disclosed hereinmay be incorporated into a polypeptide such as a full length antibody.Such an approach is demonstrated by McCoy et al Retrovirology 2014,11:83, who provide an anti-HIV VHH engineered as a fusion with a humanFc region (including hinge, CH2 and CH3 domains), expressed as a dimerconstruct.

Substituting at least one amino acid residue in the framework region ofa non human immunoglobulin variable domain with the correspondingresidue from a human variable domain is humanisation. Humanisation of avariable domain may reduce immunogenicity in humans.

Suitably, the polypeptide of the present invention consists of animmunoglobulin chain variable domain. Suitably, the polypeptide of thepresent invention is an antibody or an antibody fragment. Suitably theantibody fragment is a VHH, a VH, a VL, a V-NAR, a Fab fragment, a VL ora F(ab′)2 fragment (such as a VHH or VH, most suitably a VHH).

Specificity, Affinity, Avidity and Cross-Reactivity

Specificity refers to the number of different types of antigens orantigenic determinants to which a particular antigen-binding polypeptidecan bind. The specificity of an antigen-binding polypeptide is theability of the antigen-binding polypeptide to recognise a particularantigen as a unique molecular entity and distinguish it from another.

Affinity, represented by the equilibrium constant for the dissociationof an antigen with an antigen-binding polypeptide (Kd), is a measure ofthe binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding polypeptide: the lesser thevalue of the Kd, the stronger the binding strength between an antigenicdeterminant and the antigen-binding polypeptide (alternatively, theaffinity can also be expressed as the affinity constant (Ka), which is1/Kd). Affinity can be determined by known methods, depending on thespecific antigen of interest.

Avidity is the measure of the strength of binding between anantigen-binding polypeptide and the pertinent antigen. Avidity isrelated to both the affinity between an antigenic determinant and itsantigen binding site on the antigen-binding polypeptide and the numberof pertinent binding sites present on the antigen-binding polypeptide.

Suitably, antigen-binding polypeptides of the invention will bind with adissociation constant (Kd) of 10⁻⁶ to 10⁻¹² M, more suitably 10⁻⁷ to10⁻¹² M, more suitably 10⁻⁸ to 10⁻¹² M and more suitably 10⁻⁹ to 10⁻¹²M.

Any Kd value less than 10⁻⁶ is considered to indicate binding. Specificbinding of an antigen-binding polypeptide to an antigen or antigenicdeterminant can be determined in any suitable known manner, including,for example, Scatchard analysis and/or competitive binding assays, suchas radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwichcompetition assays, and the different variants thereof known in the art.

An anti-TNF-alpha polypeptide, a polypeptide which interacts withTNF-alpha, or a polypeptide against TNF-alpha, are all effectivelypolypeptides which bind to TNF-alpha. A polypeptide of the invention maybind to a linear or conformational epitope on TNF-alpha. The term “bindsto TNF-alpha” means binding to trimeric TNF-alpha, binding to a monomerof TNF-alpha and/or binding to a portion of a monomer of TNF-alpha.

Suitably, the polypeptide of the invention will bind to both soluble andmembrane TN F-alpha. Suitably, the polypeptide of the invention willbind to human TNF-alpha. More suitably, the polypeptide of the inventionwill bind to both human and at least one additional primate TNF-alphaselected from the group consisting of baboon TNF-alpha, marmosetTNF-alpha, cynomolgus TNF-alpha and rhesus TNF-alpha. Most suitably, thepolypeptide of the invention binds to both human and cynomolgusTNF-alpha.

Suitably, the polypeptide of the invention will neutralise both solubleand membrane TNF-alpha. Suitably, the polypeptide of the invention willneutralise human TNF-alpha. More suitably, the polypeptide of theinvention will neutralise both human and at least one additional primateTNF-alpha selected from the group consisting of baboon TNF-alpha,marmoset TNF-alpha, cynomolgus TNF-alpha and rhesus TNF-alpha. Mostsuitably, the polypeptide of the invention neutralises both human andcynomolgus TNF-alpha.

Suitably, TNF-alpha is a polypeptide comprising SEQ ID NO: 9, moresuitably TNF-alpha is a polypeptide consisting of SEQ ID NO: 9.Suitably, TNF-alpha is a polypeptide comprising SEQ ID NO: 10, moresuitably TNF-alpha is a polypeptide consisting of SEQ ID NO: 10.

Suitably, TNF-alpha is a polypeptide comprising SEQ ID NO: 11, moresuitably TNF-alpha is a polypeptide consisting of SEQ ID NO: 11.Suitably, TNF-alpha is a polypeptide comprising SEQ ID NO: 12, moresuitably TNF-alpha is a polypeptide consisting of SEQ ID NO: 12.

Polypeptides capable of reacting with TNF-alpha from humans andTNF-alpha from another species (“cross-reacting”), such as withcynomolgus monkey TNF-alpha, are advantageous because they allowpreclinical studies to be more readily performed in animal models.

Suitably the polypeptide of the invention is directed against epitopeson TNF-alpha (and in particular of the TNF-alpha trimer) that lie inand/or form part of the receptor binding site(s) of the TNF-alphatrimer, such that said polypeptide of the invention, upon binding to aTNF-alpha trimer, is capable inhibiting or reducing the TNF-alphareceptor crosslinking that is mediated by said TNF-alpha trimer and/orthe signal transduction that is mediated by such receptor crosslinking.

The polypeptides of the present invention bind to one or more epitope(s)on the TNF-alpha trimer. In one aspect of the invention there isprovided a polypeptide which binds to the same epitope on the TNF-alphatrimer as Q65F2, Q65F3, Q62F2, Q65G1, Q65H6, Q65F1, Q65D1, Q65C7, Q65D3,Q65B1, Q65F6, Q65F11, Q65E12, Q65C12, Q65A6, Q65A3, Q62E10, Q62F10,ID7F-EV, ID8F-EV, ID9F-EV, ID13F-EV, ID14F-EV, ID15F-EV, ID22F, ID23F,ID24F, ID25F, ID26F, ID27F, ID28F, ID29F, ID34F, ID37F or ID38F.

Suitably, the polypeptide of the invention is isolated. An “isolated”polypeptide is one that is removed from its original environment. Forexample, a naturally-occurring polypeptide of the invention is isolatedif it is separated from some or all of the coexisting materials in thenatural system.

Potency, Inhibition and Neutralisation

Potency is a measure of the activity of a therapeutic agent expressed interms of the amount required to produce an effect of given intensity. Ahighly potent agent evokes a greater response at low concentrationscompared to an agent of lower potency that evokes a smaller response atlow concentrations. Potency is a function of affinity and efficacy.Efficacy refers to the ability of therapeutic agent to produce abiological response upon binding to a target ligand and the quantitativemagnitude of this response. The term half maximal effectiveconcentration (EC50) refers to the concentration of a therapeutic agentwhich causes a response halfway between the baseline and maximum after aspecified exposure time. The therapeutic agent may cause inhibition orstimulation. It is commonly used, and is used herein, as a measure ofpotency.

A neutralising polypeptide for the purposes of the invention is apolypeptide which binds to TN F-alpha, inhibiting the binding ofTNF-alpha to one or both of its cognate receptors (e.g. TNFR1, TNFR2) asmeasured by ELISA. Alternatively, or in addition, a neutralisingpolypeptide for the purposes of the invention is a polypeptide whichdefends a cell from the effects of TNF-alpha by, for example, inhibitingthe biological effect of TNF-alpha. Conventionally, anti-TNF-alphatherapeutic antibody products have used an L929 murine cell line with acell death endpoint as a neutralisation assay (Humphreys and Wilson 1999Cytokine 11(10):773-782). Methods for this purpose which utilise theL929 murine cell line include the following:

Fixed-concentration L929 assay—a fixed-concentration L929 assay can beused for a relatively quick indication of the ability of a fixedconcentration of polypeptide e.g. contained within periplasmic extractto neutralise the effects of TNF-alpha cytotoxicity (as detailed in part2.2.3 of the Examples section).

Normal L929 assay—using known concentrations of anti-TNF-alphapolypeptide, a normal L929 assay can be performed (as detailed in parts3.2 to 3.2.3 of the Examples section) to assay the ability of ananti-TNF-alpha polypeptide to neutralise the effects of TNF-alphacytotoxicity by ascertaining the half maximal effective concentration(EC50) of the anti-TN F-alpha polypeptide.

Suitably the polypeptide or construct of the invention neutralizes humanTNF-alpha cytotoxicity in the normal L929 assay with an EC50 of 1 nM orless, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.7 nM orless, such as 0.6 nM or less, such as 0.5 nM or less, such as 0.4 nM orless, such as 0.3 nM or less, such as 0.2 nM or less, such as 0.1 nM orless, such as 0.09 nM or less, such as 0.08 nM or less, such as 0.07 nMor less, such as 0.06 nM or less, such as 0.05 nM or less, such as 0.04nM or less.

Suitably the polypeptide or construct of the invention neutralizescynomolgus TNF-alpha cytotoxicity in the normal L929 assay with an EC50of 1 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as0.7 nM or less, such as 0.6 nM or less, such as 0.5 nM or less, such as0.4 nM or less, such as 0.3 nM or less, such as 0.2 nM or less, such as0.1 nM or less, such as 0.09 nM or less, such as 0.08 nM or less, suchas 0.07 nM or less, such as 0.06 nM or less, such as 0.05 nM or less,such as 0.04 nM or less, such as 0.03 nM or less, such as 0.02 nM orless, such as 0.01 nM or less.

Suitably, the polypeptide of the invention inhibits binding of humanTNF-alpha to TNFR2 in an ELISA assay with an EC50 of 30 nM or less, moresuitably 10 nM or less, more suitably 3 nM or less, more suitably 1 nMor less, more suitably 0.6 nM or less, more suitably 0.5 nM or less,more suitably 0.4 nM or less, more suitably 0.3 nM or less.

Suitably, the polypeptide of the invention inhibits binding ofcynomolgus monkey TNF-alpha to TNFR2 in an ELISA assay with an EC50 of110 nM or less, more suitably 30 nM or less, more suitably 10 nM orless, more suitably 3 nM or less, more suitably 1 nM or less, moresuitably 0.6 nM or less, more suitably 0.5 nM or less, more suitably 0.4nM or less, more suitably 0.3 nM or less.

Suitably, the polypeptide of the invention inhibits binding of humanTNF-alpha to TNFR1 in an ELISA assay with an EC50 of 2 nM or less, moresuitably 1 nM or less, more suitably 0.9 nM or less, more suitably 0.8nM or less, more suitably 0.7 nM or less, more suitably 0.6 nM or less,more suitably 0.5 nM or less, more suitably 0.4 nM or less, moresuitably 0.3 nM or less.

Suitably, the polypeptide of the invention inhibits soluble humanTNF-alpha-induced HEK-293-NF-kappa-B SEAP reporter cell activation withan EC50 of 3 nM or less, suitably 2 nM or less, suitably 1 nM or less,suitably 0.5 nM or less, suitably 0.4 nM or less, suitably 0.3 nM orless, suitably 0.2 nM or less, suitably 0.1 nM or less, suitably 0.08 nMor less.

Suitably, the polypeptide of the invention inhibits membrane humanTNF-alpha-induced HEK-293-NF-kappa-B SEAP reporter cell activation withan EC50 of 300 nM or less, suitably 150 nM or less, suitably 100 nM orless, suitably 80 nM or less, suitably 40 nM or less, suitably 30 nM orless, suitably 25 nM or less, suitably 20 nM or less, suitably 15 nM orless.

Polypeptide and Polynucleotide Sequences

For the purposes of comparing two closely-related polypeptide sequences,the “% sequence identity” between a first polypeptide sequence and asecond polypeptide sequence may be calculated using NCBI BLAST v2.0,using standard settings for polypeptide sequences (BLASTP). For thepurposes of comparing two closely-related polynucleotide sequences, the“% sequence identity” between a first nucleotide sequence and a secondnucleotide sequence may be calculated using NCBI BLAST v2.0, usingstandard settings for nucleotide sequences (BLASTN).

Polypeptide or polynucleotide sequences are said to be the same as oridentical to other polypeptide or polynucleotide sequences, if theyshare 100% sequence identity over their entire length. Residues insequences are numbered from left to right, i.e. from N- to C-terminusfor polypeptides; from 5′ to 3′ terminus for polynucleotides.

A “difference” between sequences refers to an insertion, deletion orsubstitution of a single amino acid residue in a position of the secondsequence, compared to the first sequence. Two polypeptide sequences cancontain one, two or more such amino acid differences. Insertions,deletions or substitutions in a second sequence which is otherwiseidentical (100% sequence identity) to a first sequence result in reduced% sequence identity. For example, if the identical sequences are 9 aminoacid residues long, one substitution in the second sequence results in asequence identity of 88.9%. If the identical sequences are 17 amino acidresidues long, two substitutions in the second sequence results in asequence identity of 88.2%. If the identical sequences are 7 amino acidresidues long, three substitutions in the second sequence results in asequence identity of 57.1%. If first and second polypeptide sequencesare 9 amino acid residues long and share 6 identical residues, the firstand second polypeptide sequences share greater than 66% identity (thefirst and second polypeptide sequences share 66.7% identity). If firstand second polypeptide sequences are 17 amino acid residues long andshare 16 identical residues, the first and second polypeptide sequencesshare greater than 94% identity (the first and second polypeptidesequences share 94.1% identity). If first and second polypeptidesequences are 7 amino acid residues long and share 3 identical residues,the first and second polypeptide sequences share greater than 42%identity (the first and second polypeptide sequences share 42.9%identity).

Alternatively, for the purposes of comparing a first, referencepolypeptide sequence to a second, comparison polypeptide sequence, thenumber of additions, substitutions and/or deletions made to the firstsequence to produce the second sequence may be ascertained. An additionis the addition of one amino acid residue into the sequence of the firstpolypeptide (including addition at either terminus of the firstpolypeptide). A substitution is the substitution of one amino acidresidue in the sequence of the first polypeptide with one differentamino acid residue. A deletion is the deletion of one amino acid residuefrom the sequence of the first polypeptide (including deletion at eitherterminus of the first polypeptide).

For the purposes of comparing a first, reference polynucleotide sequenceto a second, comparison polynucleotide sequence, the number ofadditions, substitutions and/or deletions made to the first sequence toproduce the second sequence may be ascertained. An addition is theaddition of one nucleotide residue into the sequence of the firstpolynucleotide (including addition at either terminus of the firstpolynucleotide). A substitution is the substitution of one nucleotideresidue in the sequence of the first polynucleotide with one differentnucleotide residue. A deletion is the deletion of one nucleotide residuefrom the sequence of the first polynucleotide (including deletion ateither terminus of the first polynucleotide).

A “conservative” amino acid substitution is an amino acid substitutionin which an amino acid residue is replaced with another amino acidresidue of similar chemical structure and which is expected to havelittle influence on the function, activity or other biologicalproperties of the polypeptide. Such conservative substitutions suitablyare substitutions in which one amino acid within the following groups issubstituted by another amino acid residue from within the same group:

Amino acid Group residue Non-polar Glycine aliphatic Alanine ValineLeucine Isoleucine Aromatic Phenylalanine Tyrosine Tryptophan PolarSerine uncharged Threonine Asparagine Glutamine Negatively Aspartatecharged Glutamate Positively Lysine charged Arginine

Suitably, a hydrophobic amino acid residue is a non-polar amino acid.More suitably, a hydrophobic amino acid residue is selected from V, I,L, M, F, W or C.

As used herein, numbering of polypeptide sequences and definitions ofCDRs and FRs are as defined according to the Kabat system (Kabat et al1991 Sequences of Proteins of Immunological Interest, Fifth Edition U.S.Department of Health and Human Services, NIH Publication Number 91-3242,herein incorporated by reference in its entirety). A “corresponding”amino acid residue between a first and second polypeptide sequence is anamino acid residue in a first sequence which shares the same positionaccording to the Kabat system with an amino acid residue in a secondsequence, whilst the amino acid residue in the second sequence maydiffer in identity from the first. Suitably corresponding residues willshare the same number (and letter) if the framework and CDRs are thesame length according to Kabat definition. Alignment can be achievedmanually or by using, for example, a known computer algorithm forsequence alignment such as NCBI BLAST v2.0 (BLASTP or BLASTN) usingstandard settings.

Suitably, the polynucleotides used in the present invention areisolated. An “isolated” polynucleotide is one that is removed from itsoriginal environment. For example, a naturally-occurring polynucleotideis isolated if it is separated from some or all of the coexistingmaterials in the natural system. A polynucleotide is considered to beisolated if, for example, it is cloned into a vector that is not a partof its natural environment or if it is comprised within cDNA.

In one aspect of the invention there is provided a polynucleotideencoding the polypeptide or construct of the invention. Suitably thepolynucleotide comprises or consists of a sequence sharing 70% orgreater, such as 80% or greater, such as 90% or greater, such as 95% orgreater, such as 99% or greater sequence identity with any one of SEQ IDNOs: 83 to 88.

More suitably the polynucleotide comprises or consists of any one of SEQID NOs: 83 to 88. In a further aspect there is provided a cDNAcomprising said polynucleotide.

In one aspect of the invention there is provided a polynucleotidecomprising or consisting of a sequence sharing 70% or greater, such as80% or greater, such as 90% or greater, such as 95% or greater, such as99% or greater sequence identity with any one of the portions of any oneof SEQ ID NOs: 83 to 86 which encodes CDR1, CDR2 or CDR3 of the encodedimmunoglobulin chain variable domain.

Suitably, the polypeptide sequence of the present invention contains atleast one alteration with respect to a native sequence. Suitably, thepolynucleotide sequences of the present invention contain at least onealteration with respect to a native sequence. Suitably the alteration tothe polypeptide sequence or polynucleotide sequence is made to increasestability of the polypeptide or encoded polypeptide to proteases presentin the intestinal tract (for example trypsin and chymotrypsin).

The Kabat numbering system applied to selected immunoglobulin chainvariable domain sequences

Region FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 Residue # 1 2 3 45 6 7 8 9 10 11 12 Q62E10 Q V Q L V E S G G G L V Q6561 E V Q L V E S GG G L V ID38F D V Q L V E S G G G L V TNF1 E V Q L V E S G G G L V KabatH1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 numbering Region FR1 FR1 FR1 FR1FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 Residue # 13 14 15 16 17 18 19 20 21 2223 24 Q62E10 Q P G G S L R L S C T T Q6561 Q P G G S L K L S C A A ID38FQ P G G S L K L S C A A TNF1 Q P G G S L R L S C A A Kabat H13A H14 H15H16 H17 H18 H19 H20 H21 H22 H23 H24 numbering Region FR1 FR1 FR1 FR1 FR1FR1 CDR1 CDR1 CDR1 CDR1 CDR1 FR2 Residue # 25 26 27 28 29 30 31 32 33 3435 36 Q62E10 S G L D F G I H W M Y W Q6561 S G F D F S S H W M Y W ID38FS G F D F S S H W M Y W TNF1 S G F T F S D Y W M Y W Kabat H25 H26 H27H28 H29 H30 H31 H32 H33 H34 H35 H36 numbering Region FR2 FR2 FR2 FR2 FR2FR2 FR2 FR2 FR2 CDR2 FR2 FR2 Residue # 37 38 39 40 41 42 43 44 45 46 4748 Q62E10 F R Q A P G K E L E W V Q6561 V R Q A P G K E L E W L ID38F VR Q A P G K E L E W L TNF1 V R Q A P G K G L E W V Kabat H37 H38 H39 H40H41 H42 H43 H44 H45 H46 H47 H48 numbering Region FR2 CDR2 CDR2 CDR2 CDR2CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 Residue # 49 50 51 52 53 54 55 56 5758 59 60 Q62E10 A E I N T N A L I T K Y Q6561 S E I N T N G L I T K YID38F S E I N T N G L I T H Y TNF1 S E I N T N G L I T K Y Kabat H49 H50H51 H52 H52A H53 H54 H55 H56 H57 H58 H59 numbering Region CDR2 CDR2 CDR2CDR2 CDR2 CDR2 FR3 FR3 FR3 FR3 FR3 FR3 Residue # 61 62 63 64 65 66 67 6869 70 71 72 Q62E10 A D S V K G R F T I S R Q6561 G D S V K G R F T V S RID38F G D S V K G R F T V S R TNF1 P D S V K G R F T I S R Kabat H60 H61H62 H63 H64 H65 H66 H67 H68 H69 H70 H71 numbering Region FR3 FR3 FR3 FR3FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 Residue # 73 74 75 76 77 78 79 80 81 8283 84 Q62E10 D N A K N T L F L Q M N Q6561 N N A A N K M Y L E L T ID38FN N A A N K M Y L E L T TNF1 D N A K N T L Y L Q M N Kabat H72 H73 H74H75 H76 H77 H78 H79 H80 H81 H82 H82A numbering Region FR3 FR3 FR3 FR3FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 Residue # 85 86 87 88 89 90 91 92 93 9495 96 Q62E10 D L K S E D T A V Y Y C Q6561 R L E P E D T A L Y Y C ID38FR L E P E D T A L Y Y C TNF1 S L K P E D T A L Y Y C Kabat H82B H82C H83H84 H85 H86 H87 H88 H89 H90 H91 H92 numbering Region FR3 FR3 CDR3 CDR3CDR3 CDR3 CDR3 CDR3 FR4 FR4 FR4 FR4 Residue # 97 98 99 100 101 102 103104 105 106 105 106 Q62E10 S N T Q N G A A K G K G Q6561 A R N Q K G L NK G K G ID38F A R N Q H G L N K G K G TNF1 A R S P S G F N R G R G KabatH93 H94 H95 H96 H97 H98 H101 H102 H103 H104 H103 H104 numbering RegionFR4 FR4 FR4 FR4 FR4 FR4 FR4 FR4 FR4 Residue # 107 108 109 110 111 112113 114 115 Q62E10 Q G V Q V T V S S Q6561 Q G T Q V T V S S ID38F Q G TQ V T V S S TNF1 Q G T Q V T V S S Kabat H105 H106 H107 H108 H109 H110H111 H112 H113 numbering

Sequences Belonging to Family 1

Suitably CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 80% or greater sequenceidentity with SEQ ID NO: 1.

Alternatively, CDR1 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 2,more suitably no more than 1 addition(s) compared to SEQ ID NO: 1.Suitably, CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 2, moresuitably no more than 1 substitution(s) compared to SEQ ID NO: 1.Suitably, CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 2, moresuitably no more than 1 deletion(s) compared to SEQ ID NO: 1.

Suitably any residues of CDR1 differing from their correspondingresidues in SEQ ID NO: 1 are conservative substitutions with respect totheir corresponding residues. Suitably CDR1 comprises or more suitablyconsists of SEQ ID NO: 1. Suitably the sequence of CDR1 is SEQ ID NO: 1,SEQ ID NO: 59 or SEQ ID NO: 60.

Suitably CDR2 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 55%, 60%, 70%, 75%, 80%,85%, 90% or greater sequence identity, with SEQ ID NO: 2.

Alternatively, CDR2 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 8,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1addition(s) compared to SEQ ID NO: 2. Suitably, CDR2 of the polypeptideof the present invention comprises or more suitably consists of asequence having no more than 8, more suitably no more than 7, moresuitably no more than 6, more suitably no more than 5, more suitably nomore than 4, more suitably no more than 3, more suitably no more than 2,more suitably no more than 1 substitution(s) compared to SEQ ID NO: 2.Suitably, CDR2 of the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 8, moresuitably no more than 7, more suitably no more than 6, more suitably nomore than 5, more suitably no more than 4, more suitably no more than 3,more suitably no more than 2, more suitably no more than 1 deletion(s)compared to SEQ ID NO: 2.

Suitably any residues of CDR2 differing from their correspondingresidues in SEQ ID NO: 2 are conservative substitutions with respect totheir corresponding residues. Suitably the residue of CDR2 correspondingto residue number 10 of SEQ ID NO: 2 is R, H, D, E, N, Q, S, T, Y, G, A,V, L, W, P, M, C, F or I (most suitably H). Suitably CDR2 comprises ormore suitably consists of SEQ ID NO: 2. Suitably the sequence of CDR2 isSEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 2, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69.

Suitably CDR3 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 80% or greater sequenceidentity with SEQ ID NO: 3.

Alternatively, CDR3 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 3,more suitably no more than 2, more suitably no more than 1 addition(s)compared to SEQ ID NO: 3. Suitably, CDR3 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 3, more suitably no more than 2, more suitably nomore than 1 substitution(s) compared to SEQ ID NO: 3. Suitably, CDR3 ofthe polypeptide of the present invention comprises or more suitablyconsists of a sequence having no more than 3, more suitably no more than2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 3.Suitably, any substitutions are conservative, with respect to theircorresponding residues in SEQ ID NO: 3.

Suitably any residues of CDR3 differing from their correspondingresidues in SEQ ID NO: 3 are conservative substitutions with respect totheir corresponding residues. Suitably the residue of CDR3 correspondingto residue number 3 of SEQ ID NO: 3 is R, H, D, E, N, Q, S, T, Y, G, A,V, L, W, P, M, C, F or I; or suitably R, H, D, E, N, Q, S, T, Y, G, V,L, W, P, M, C, F or I (most suitably H). Suitably the residue of CDR3corresponding to residue number 3 of SEQ ID NO: 3 is H and any otherresidues of CDR3 differing from their corresponding residues in SEQ IDNO: 3 are conservative substitutions with respect to their correspondingresidues. Suitably CDR3 comprises or more suitably consists of SEQ IDNO: 3.

Alternatively CDR3 of the polypeptide of the present invention comprisesor more suitably consists of a sequence sharing 50%, such as 60%, suchas 80% or greater sequence identity with SEQ ID NO: 3 and whereinresidue number 3 of SEQ ID NO: 3 is R, D, N, C, E, Q, G, H, I, L, K, M,F, P, S, T, W, Y or V (suitably H or a conservative substitution of H;more suitably H). Alternatively residue number 3 of SEQ ID NO: 3 is H ora conservative substitution of H (most suitably H) and any otherresidues of CDR3 differing from their corresponding residues in SEQ IDNO: 3 are conservative substitutions. Suitably the sequence of CDR3 isSEQ ID NO: 3, SEQ ID NO: 70, SEQ ID NO: 71 or SEQ ID NO: 72

Suitably residue 1 of CDR1 is S, V or N; residues 2 to 4 are HWM andresidue 5 is Y or C. Suitably, residues 1 to 9 of CDR2 are EINTNGLIT;residue 10 is H, K, S or N; residue 11 is Y, residue 12 is G, V, I or A;residue 13 is D; residue 14 is S or F; residue 15 is V or T; residue 16is H, K, R or G and residue 17 is G. Suitably residue 1 of CDR3 is N;residue 2 is Q or E; residue 3 is H, K, M or R and residues 4 to 6 areGLN.

Some particularly suitable CDR sequences are shown in the table below.Suitably, CDR1 of the polypeptide of the invention is one of the CDR1sequences listed below. Suitably, CDR2 of the polypeptide of theinvention is one of the CDR2 sequences listed below. Suitably, CDR3 ofthe polypeptide of the invention is one of the CDR3 sequences listedbelow. Suitably, the polypeptide of the invention comprises acombination of two, or more suitably three, of the CDR sequences listedbelow.

Particular Family 1 CDRs of the polypeptide of the invention:

CDR1 CDR2 CDR3 SHWMY EINTNGLITHYGDSVHG NQHGLN (SEQ ID NO: 1)(SEQ ID NO: 61) (SEQ ID NO: 3) VHWMY EINTNGLITKYGDSVHG NQKGLN(SEQ ID NO: 59) (SEQ ID NO: 62) (SEQ ID NO: 70) NHWMC EINTNGLITHYGDSVKGNQMGLN (SEQ ID NO: 60) (SEQ ID NO: 2) (SEQ ID NO: 71) EINTNGLITSYVDSVKGNERGLN (SEQ ID NO: 63) (SEQ ID NO: 72) EINTNGLITKYIDSVRG (SEQ ID NO: 64)EINTNGLITNYVDSVKG (SEQ ID NO: 65) EINTNGLITKYIDSVGG (SEQ ID NO: 66)EINTNGLITKYADFVKG (SEQ ID NO: 67) EINTNGLITKYADSTKG (SEQ ID NO: 68)EINTNGLITKYGDSVKG (SEQ ID NO: 69)

Percentage identity of CDRs of ID38F to other Family 1 members, TNF1 andQ62F11 Name CDR1 CDR2 CDR3 Q65B1 100 94.1 83.3 Q65F2 100 88.2 83.3 Q65F3100 82.4 83.3 Q62F2 100 88.2 83.3 Q65G1 100 82.4 83.3 Q65H6 100 82.483.3 Q65F1 80 82.4 83.3 Q65D1 60 82.4 83.3 Q65D3 100 82.4 66.7 Q65C7 10082.4 83.3 TNF1 60 88.2 33.3 Q62F11 20 52.9 13.3

Suitably FR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 5%, 12%, 18%, 26%, 32%,38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% orgreater sequence identity, with SEQ ID NO: 4.

Alternatively, FR1 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 28, moresuitably no more than 26, more suitably no more than 24, more suitablyno more than 22, more suitably no more than 20, more suitably no morethan 18, more suitably no more than 16, more suitably no more than 14,more suitably no more than 13, more suitably no more than 12, moresuitably no more than 11, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 addition(s) compared to SEQ IDNO: 4. Suitably, FR1 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than28, more suitably no more than 26, more suitably no more than 24, moresuitably no more than 22, more suitably no more than 20, more suitablyno more than 18, more suitably no more than 16, more suitably no morethan 14, more suitably no more than 13, more suitably no more than 12,more suitably no more than 11, more suitably no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 substitution(s)compared to SEQ ID NO: 4. Suitably, FR1 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 28, more suitably no more than 26, more suitably nomore than 24, more suitably no more than 22, more suitably no more than20, more suitably no more than 18, more suitably no more than 16, moresuitably no more than 14, more suitably no more than 13, more suitablyno more than 12, more suitably no more than 11, more suitably no morethan 10, more suitably no more than 9, more suitably no more than 8,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1deletion(s) compared to SEQ ID NO: 4.

Suitably any residues of FR1 differing from their corresponding residuesin SEQ ID NO: 4 are conservative substitutions with respect to theircorresponding residues. Suitably the residue of FR1 corresponding toresidue number 1 of SEQ ID NO: 4 is G, A, V, L, I, F, P, S, T, Y, C, M,K, R, H, W, D, E or N (more suitably D or E, most suitably D). Suitablythe residue of FR1 corresponding to residue number 5 of SEQ ID NO: 4 isG, A, V, L, I, F, P, S, T, Y, C, M, K, R, H, W, D, E or N (suitably V).Suitably the residues of FR1 corresponding to residue numbers 1 to 5 ofSEQ ID NO: 4 are DVQLV. Suitably the residue of FR1 corresponding toresidue numbers 20 and/or 24 of SEQ ID NO: 4 are an amino acid which ishydrophobic (most suitably L or A, respectively). Suitably the residueof FR1 corresponding to residue number 29 of SEQ ID NO: 4 is F. SuitablyFR1 comprises or more suitably consists of SEQ ID NO: 4.

Suitably FR2 of the polypeptide of the present invention comprises ormore suitably consists of 5 a sequence sharing 10%, 15%, 25%, 30%, 40%,45%, 55%, 60%, 70%, 75%, 85%, 90% or greater sequence identity, with SEQID NO: 5.

Alternatively, FR2 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 13, moresuitably no more than 12, more suitably no more than 11, more suitablyno more than 10, more suitably no more than 9, more suitably no morethan 8, more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1addition(s) compared to SEQ ID NO: 5. Suitably, FR2 of the polypeptideof the present invention comprises or more suitably consists of asequence having no more than 13, more suitably no more than 12, moresuitably no more than 11, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 substitution(s) compared toSEQ ID NO: 5. Suitably, FR2 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than13, more suitably no more than 12, more suitably no more than 11, moresuitably no more than 10, more suitably no more than 9, more suitably nomore than 8, more suitably no more than 7, more suitably no more than 6,more suitably no more than 5, more suitably no more than 4, moresuitably no more than 3, more suitably no more than 2, more suitably nomore than 1 deletion(s) compared to SEQ ID NO: 5.

Suitably any residues of FR2 differing from their corresponding residuesin SEQ ID NO: 5 are conservative substitutions with respect to theircorresponding residues. Suitably the residues of FR2 corresponding toresidue numbers 8 to 11 of SEQ ID NO: 5 are KEXE, wherein X is R or L.Alternatively the residues of FR2 corresponding to residue numbers 9 to12 of SEQ ID NO: 5 are GLEW. Suitably FR2 comprises or more suitablyconsists of SEQ ID NO: 5.

Suitably FR3 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 8%, 15%, 20%, 26%, 32%,40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% orgreater sequence identity, with SEQ ID NO: 6.

Alternatively, FR3 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 29, moresuitably no more than 27, more suitably no more than 25, more suitablyno more than 23, more suitably no more than 21, more suitably no morethan 19, more suitably no more than 17, more suitably no more than 15,more suitably no more than 13, more suitably no more than 11, moresuitably no more than 9, more suitably no more than 7, more suitably nomore than 6, more suitably no more than 5, more suitably no more than 4,more suitably no more than 3, more suitably no more than 2, moresuitably no more than 1 addition(s) compared to SEQ ID NO: 6. Suitably,FR3 of the polypeptide of the present invention comprises or moresuitably consists of a sequence having no more than 29, more suitably nomore than 27, more suitably no more than 25, more suitably no more than23, more suitably no more than 21, more suitably no more than 19, moresuitably no more than 17, more suitably no more than 15, more suitablyno more than 13, more suitably no more than 11, more suitably no morethan 9, more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1substitution(s) compared to SEQ ID NO: 6. Suitably, FR3 of thepolypeptide of the present invention comprises or more suitably consistsof a sequence having no more than 29, more suitably no more than 27,more suitably no more than 25, more suitably no more than 23, moresuitably no more than 21, more suitably no more than 19, more suitablyno more than 17, more suitably no more than 15, more suitably no morethan 13, more suitably no more than 11, more suitably no more than 9,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1deletion(s) compared to SEQ ID NO: 6.

Suitably the residue of FR3 corresponding to residue number 26 of SEQ IDNO: 6 is an amino acid which is hydrophobic (suitably A). Suitably anyresidues of FR3 differing from their corresponding residues in SEQ IDNO: 6 are conservative substitutions with respect to their correspondingresidues. Suitably FR3 comprises or more suitably consists of SEQ ID NO:6.

Suitably FR4 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or greater sequence identity, with SEQ ID NO: 7.

Alternatively, FR4 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 addition(s)compared to SEQ ID NO: 7. Suitably, FR4 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 10, more suitably no more than 9, more suitably nomore than 8, more suitably no more than 7, more suitably no more than 6,more suitably no more than 5, more suitably no more than 4, moresuitably no more than 3, more suitably no more than 2, more suitably nomore than 1 substitution(s) compared to SEQ ID NO: 7. Suitably, FR4 ofthe polypeptide of the present invention comprises or more suitablyconsists of a sequence having no more than 10, more suitably no morethan 9, more suitably no more than 8, more suitably no more than 7, moresuitably no more than 6, more suitably no more than 5, more suitably nomore than 4, more suitably no more than 3, more suitably no more than 2,more suitably no more than 1 deletion(s) compared to SEQ ID NO: 7.

Suitably any residues of FR4 differing from their corresponding residuesin SEQ ID NO: 7 are conservative substitutions with respect to theircorresponding residues. Suitably FR4 comprises or more suitably consistsof SEQ ID NO: 7.

Suitably the polypeptide of the present invention comprises or moresuitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity,with SEQ ID NO: 8.

Alternatively, the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 20, moresuitably no more than 15, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 addition(s) compared to SEQ IDNO: 8. Suitably, the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 20, moresuitably no more than 15, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 substitution(s) compared toSEQ ID NO: 8. Suitably, the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than20, more suitably no more than 15, more suitably no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 deletion(s)compared to SEQ ID NO: 8.

Suitably the N-terminus of the polypeptide is D. Suitably thepolypeptide comprises or more suitably consists of SEQ ID NO: 8.

Sequences Belonging to Family 2

Suitably CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 80% or greater sequenceidentity with SEQ ID NO: 15.

Alternatively, CDR1 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 2,more suitably no more than 1, addition(s), compared to SEQ ID NO: 15.Suitably, CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 2, moresuitably no more than 1, substitution(s) compared to SEQ ID NO: 15.Suitably, CDR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 2, moresuitably no more than 1, deletion(s) compared to SEQ ID NO: 15.

Suitably any residues of CDR1 differing from their correspondingresidues in SEQ ID NO: 15 are conservative substitutions with respect totheir corresponding residues. Suitably CDR1 comprises or more suitablyconsists of SEQ ID NO: 15.

Suitably CDR2 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 55%, 60%, 70%, 75%, 80%,85%, 90% or greater sequence identity, with SEQ ID NO: 16.

Alternatively, CDR2 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 8,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1,addition(s) compared to SEQ ID NO: 16. Alternatively, CDR2 of thepolypeptide of the present invention comprises or more suitably consistsof a sequence having no more than 8, more suitably no more than 7, moresuitably no more than 6, more suitably no more than 5, more suitably nomore than 4, more suitably no more than 3, more suitably no more than 2,more suitably no more than 1, substitutions(s) compared to SEQ ID NO:16. Alternatively, CDR2 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 8,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1,deletions(s) compared to SEQ ID NO: 16.

Suitably the residue of CDR2 corresponding to residue number 10 and/or16 of SEQ ID NO: 16 is R, H, D, E, N, Q, S, T, Y, G, A, V, L, W, P, M,C, F or I (more suitably H). Suitably any residues of CDR2 differingfrom their corresponding residues in SEQ ID NO: 16 are conservativesubstitutions with respect to their corresponding residues. SuitablyCDR2 comprises or more suitably consists of SEQ ID NO: 16. Suitably thesequence of CDR2 is SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77 or SEQ ID NO: 16.

Suitably CDR3 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 60% or greater sequenceidentity, such as 80% or greater sequence identity, with SEQ ID NO: 17.

Alternatively, CDR3 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than 3,more suitably no more than 2, more suitably no more than 1, addition(s)compared to SEQ ID NO: 17. Suitably, CDR3 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 3, more suitably no more than 2, more suitably nomore than 1, substitution(s) compared to SEQ ID NO: 17. Suitably, CDR3of the polypeptide of the present invention comprises or more suitablyconsists of a sequence having no more than 3, more suitably no more than2, more suitably no more than 1, deletion(s) compared to SEQ ID NO: 17.

Suitably any residues of CDR3 differing from their correspondingresidues in SEQ ID NO: 17 are conservative substitutions with respect totheir corresponding residues. Suitably the sequence of CDR3 is SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 17 or SEQ ID NO: 80. Suitably CDR3comprises or more suitably consists of SEQ ID NO: 17.

Suitably residues 1 to 5 of CDR1 are IHWMY. Suitably residues 1 to 9 ofCDR2 are EINTNGLIT; residue 10 is L, T, H, K or V; residue 11 is Y;residue 12 is S, A, T or P; residues 13 to 15 are DSV; residue 16 is R,K or S and residue 17 is G. Suitably residue 1 of CDR3 is S, A or T;residue 2 is R or Q; residues 3 to 4 are NG; residue 5 is A or K andresidue 6 is A or T.

Some particularly suitable CDR sequences are shown in the table below.Suitably, CDR1 of the polypeptide of the invention is one of the CDR1sequences listed below. Suitably, CDR2 of the polypeptide of theinvention is one of the CDR2 sequences listed below. Suitably, CDR3 ofthe polypeptide of the invention is one of the CDR3 sequences listedbelow. Suitably, the polypeptide of the invention comprises acombination of two, or more suitably three, of the CDR sequences listedbelow.

Particular Family 2 CDRs of the polypeptide of the invention:

CDR1 CDR2 CDR3 IHWMY EINTNGLITLYSDSVRG SRNGAA (SEQ ID NO: 15)(SEQ ID NO: 73) (SEQ ID NO: 78) EINTNGLITLYADSVKG ARNGAA (SEQ ID NO: 74)(SEQ ID NO: 79) EINTNALITTYADSVKG TQNGAA (SEQ ID NO: 75) (SEQ ID NO: 17)EINTNGLITHYTDSVSG TQNGKT (SED ID NO: 76) (SEQ ID NO: 80)EINTNALITKYADSVKG (SEQ ID NO: 77) EINTNGLITVYPDSVKG (SEQ ID NO: 16)

Percentage identity of CDRs of 62E10 to other Family 2 members, TNF1 andQ62F11 Name CDR 1 CDR 2 CDR 3 Q65F6 100 76.5 66.7 Q65F11 100 88.2 66.7Q65E12 100 88.2 66.7 Q62C12 100 94.1 66.7 Q65A6 100 76.5 66.7 Q65A3 100100 100 Q65F10 100 82.4 66.7 TNF1 60 88.2 16.7 Q62F11 20 52.9 13.3

Suitably FR1 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 5%, 12%, 18%, 26%, 32%,38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% orgreater sequence identity, with SEQ ID NO: 18.

Alternatively, FR1 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 28, moresuitably no more than 26, more suitably no more than 24, more suitablyno more than 22, more suitably no more than 20, more suitably no morethan 18, more suitably no more than 16, more suitably no more than 14,more suitably no more than 13, more suitably no more than 12, moresuitably no more than 11, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 addition(s) compared to SEQ IDNO: 18. Alternatively, FR1 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than28, more suitably no more than 26, more suitably no more than 24, moresuitably no more than 22, more suitably no more than 20, more suitablyno more than 18, more suitably no more than 16, more suitably no morethan 14, more suitably no more than 13, more suitably no more than 12,more suitably no more than 11, more suitably no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 substitution(s)compared to SEQ ID NO: 18. Alternatively, FR1 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 28, more suitably no more than 26, more suitably nomore than 24, more suitably no more than 22, more suitably no more than20, more suitably no more than 18, more suitably no more than 16, moresuitably no more than 14, more suitably no more than 13, more suitablyno more than 12, more suitably no more than 11, more suitably no morethan 10, more suitably no more than 9, more suitably no more than 8,more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1deletion(s) compared to SEQ ID NO: 18.

Suitably the residue of FR1 corresponding to residue number 1 of SEQ IDNO: 4 is G, A, V, L, I, F, P, S, T, Y, C, M, K, R, H, W, D, E or N (moresuitably D or E, most suitably D). Suitably the residue of FR1corresponding to residue number 5 of SEQ ID NO: 18 is G, A, V, L, I, F,P, S, T, Y, C, M, K, R, H, W, D, E or N (suitably V). Suitably theresidues of FR1 corresponding to residue numbers 1 to 5 of SEQ ID NO: 18are DVQLV. Suitably the residue of FR1 corresponding to residue number20 of SEQ ID NO: 18 is an amino acid which is hydrophobic (suitably L).Suitably the residue of FR1 corresponding to residue number 29 of SEQ IDNO:18 is F. Suitably any residues of FR1 differing from theircorresponding residues in SEQ ID NO: 18 are conservative substitutionswith respect to their corresponding residues. Suitably FR1 comprises ormore suitably consists of SEQ ID NO: 18.

Suitably FR2 of the polypeptide of the present invention comprises ormore suitably consists of a sequence sharing 10%, 15%, 25%, 30%, 40%,45%, 55%, 60%, 70%, 75%, 85%, 90% or greater sequence identity, with SEQID NO: 19.

Alternatively, FR2 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 13, moresuitably no more than 12, more suitably no more than 11, more suitablyno more than 10, more suitably no more than 9, more suitably no morethan 8, more suitably no more than 7, more suitably no more than 6, moresuitably no more than 5, more suitably no more than 4, more suitably nomore than 3, more suitably no more than 2, more suitably no more than 1addition(s) compared to SEQ ID NO: 19. Suitably, FR2 of the polypeptideof the present invention comprises or more suitably consists of asequence having no more than 13, more suitably no more than 12, moresuitably no more than 11, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 substitution(s) compared toSEQ ID NO: 19. Suitably, FR2 of the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than13, more suitably no more than 12, more suitably no more than 11, moresuitably no more than 10, more suitably no more than 9, more suitably nomore than 8, more suitably no more than 7, more suitably no more than 6,more suitably no more than 5, more suitably no more than 4, moresuitably no more than 3, more suitably no more than 2, more suitably nomore than 1 deletion(s) compared to SEQ ID NO: 19.

Suitably the residues of FR2 corresponding to residue numbers 8 to 11 ofSEQ ID NO: 19 are KELE. Suitably any residues of FR2 differing fromtheir corresponding residues in SEQ ID NO:19 are conservativesubstitutions with respect to their corresponding residues. Suitably FR2comprises or more suitably consists of SEQ ID NO: 19.

Suitably FR3 of the polypeptide of the present invention comprises asequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%,76%, 80%, 82%, 85%, 90%, 92%, 95% or greater sequence identity, with SEQID NO: 20.

Alternatively, FR3 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 29, moresuitably 27, more suitably 25, more suitably 23, more suitably 21, moresuitably 19, more suitably 17, more suitably 15, more suitably 13, moresuitably 11, more suitably 9, more suitably 7, more suitably 6, moresuitably 5, more suitably 4, more suitably 3, more suitably 2, moresuitably 1 addition(s) compared to SEQ ID NO: 20. Suitably, FR3 of thepolypeptide of the present invention comprises or more suitably consistsof a sequence having no more than 29, more suitably 27, more suitably25, more suitably 23, more suitably 21, more suitably 19, more suitably17, more suitably 15, more suitably 13, more suitably 11, more suitably9, more suitably 7, more suitably 6, more suitably 5, more suitably 4,more suitably 3, more suitably 2, more suitably 1 substitution(s)compared to SEQ ID NO: 20. Suitably, FR3 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 29, more suitably 27, more suitably 25, moresuitably 23, more suitably 21, more suitably 19, more suitably 17, moresuitably 15, more suitably 13, more suitably 11, more suitably 9, moresuitably 7, more suitably 6, more suitably 5, more suitably 4, moresuitably 3, more suitably 2, more suitably 1 deletion(s) compared to SEQID NO: 20.

Suitably the residue of FR3 corresponding to residue number 26 of SEQ IDNO: 20 is an amino acid which is hydrophobic (suitably A). Suitably anyresidues of FR3 differing from their corresponding residues in SEQ IDNO: 20 are conservative substitutions with respect to theircorresponding residues. Suitably FR3 comprises or more suitably consistsof SEQ ID NO: 20.

Suitably FR4 of the polypeptide of the present invention comprises asequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater sequence identity, with SEQ ID NO: 21.

Alternatively, FR4 of the polypeptide of the present invention comprisesor more suitably consists of a sequence having no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 addition(s)compared to SEQ ID NO: 21. Suitably, FR4 of the polypeptide of thepresent invention comprises or more suitably consists of a sequencehaving no more than 10, more suitably no more than 9, more suitably nomore than 8, more suitably no more than 7, more suitably no more than 6,more suitably no more than 5, more suitably no more than 4, moresuitably no more than 3, more suitably no more than 2, more suitably nomore than 1 substitution(s) compared to SEQ ID NO: 21. Suitably, FR4 ofthe polypeptide of the present invention comprises or more suitablyconsists of a sequence having no more than 10, more suitably no morethan 9, more suitably no more than 8, more suitably no more than 7, moresuitably no more than 6, more suitably no more than 5, more suitably nomore than 4, more suitably no more than 3, more suitably no more than 2,more suitably no more than 1 deletion(s) compared to SEQ ID NO: 21.

Suitably the residue of CDR2 corresponding to residue number 1 of SEQ IDNO: 21 is R, H, D, E, N, Q, S, T, Y, G, A, V, L, W, P, M, C, F or I(suitably H). Suitably any residues of FR4 differing from theircorresponding residues in SEQ ID NO: 21 are conservative substitutionswith respect to their corresponding residues. Suitably FR4 comprises ormore suitably consists of SEQ ID NO: 21.

Suitably the polypeptide of the present invention comprises or moresuitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity,with SEQ ID NO: 22.

Alternatively, the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 20, moresuitably no more than 15, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 addition(s) compared to SEQ IDNO: 22. Suitably, the polypeptide of the present invention comprises ormore suitably consists of a sequence having no more than 20, moresuitably no more than 15, more suitably no more than 10, more suitablyno more than 9, more suitably no more than 8, more suitably no more than7, more suitably no more than 6, more suitably no more than 5, moresuitably no more than 4, more suitably no more than 3, more suitably nomore than 2, more suitably no more than 1 substitutions(s) compared toSEQ ID NO: 22. Suitably, the polypeptide of the present inventioncomprises or more suitably consists of a sequence having no more than20, more suitably no more than 15, more suitably no more than 10, moresuitably no more than 9, more suitably no more than 8, more suitably nomore than 7, more suitably no more than 6, more suitably no more than 5,more suitably no more than 4, more suitably no more than 3, moresuitably no more than 2, more suitably no more than 1 deletion(s)compared to SEQ ID NO: 22.

Suitably the polypeptide comprises or more suitably consists of SEQ IDNO: 22. Suitably the N-terminus of the polypeptide is D. Suitably thepolypeptide comprises or more suitably consists of SEQ ID NO: 55.

Suitably, the polypeptide of the present invention comprises, or moresuitably consists of, a sequence having no more than 20, more suitablyno more than 15, more suitably no more than 10, more suitably no morethan 9, more suitably no more than 8, more suitably no more than 7, moresuitably no more than 6, more suitably no more than 5, more suitably nomore than 4, more suitably no more than 3, more suitably no more than 2,more suitably no more than 1, substitution(s) with respect to SEQ ID NO:22. Suitably, any substitutions are conservative, with respect to theircorresponding residues in SEQ ID NO: 22.

Linkers and Multimers

A construct according to the invention comprises multiple polypeptidesand therefore may suitably be multivalent. Such a construct may compriseat least two identical polypeptides according to the invention. Aconstruct consisting of two identical polypeptides according to theinvention is a “homobihead”. In one aspect of the invention there isprovided a construct comprising two or more identical polypeptides ofthe invention.

Alternatively, a construct may comprise at least two polypeptides whichare different, but are both still polypeptides according to theinvention (a “heterobihead”).

Alternatively, such a construct may comprise (a) at least onepolypeptide according to the invention and (b) at least one polypeptidesuch as an antibody or antigen-binding fragment thereof, which is not apolypeptide of the invention (also a “heterobihead”). The at least onepolypeptide of (b) may bind TNF-alpha (for example via a differentepitope to that of (a)), or alternatively may bind to a target otherthan TNF-alpha. Suitably the different polypeptide (b) binds to, forexample: an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23),an interleukin receptor (such as IL-6R and IL-7R), a transcriptionfactor (such as NF-kB), a cytokine (such as TNF-alpha, IFN-gammaTGF-beta and TSLP), a transmembrane protein (such as gp130 and CD3), asurface glycoprotein (such as CD4, CD20, CD40), a soluble protein (suchas CD40L), an integrin (such as a4b7 and AlphaEbeta7), an adhesionmolecule (such as MAdCAM), a chemokine (such as IP10 and CCL20), achemokine receptor (such as CCR2 and CCR9), an inhibitory protein (suchas SMAD7), a kinase (such as JAK3), a G protein-coupled receptor (suchas sphingosine-1-P receptor), other inflammatory mediators orimmunologically relevant ligands involved in human pathologicalprocesses. Thus the different polypeptide (b) binds to, for example,IL-6R, IL-6, IL-12, IL-23, IL-1-beta, IL-17A or CD3; or otherinflammatory mediators or immunologically relevant ligands involved inhuman pathological processes.

Constructs can be multivalent and/or multispecific. A multivalentconstruct (such as a bivalent construct) comprises two or more bindingpolypeptides therefore presents two or more sites at which attachment toone or more antigens can occur. An example of a multivalent constructcould be a homobihead or a heterobihead. A multispecific construct (suchas a bispecific construct) comprises two or more different bindingpolypeptides which present two or more sites at which either (a)attachment to two or more different antigens can occur or (b) attachmentto two or more different epitopes on the same antigen can occur. Anexample of a multispecific construct could be a heterobihead. Amultispecific construct is multivalent.

Suitably, the polypeptides comprised within the construct are antibodyfragments. More suitably, the polypeptides comprised within theconstruct are selected from the list consisting of: a VHH, a VH, a VL, aV-NAR, a Fab fragment and a F(ab′)2 fragment. More suitably, thepolypeptides comprised within the construct are VHHs.

The polypeptides of the invention can be linked to each other directly(i.e. without use of a linker) or via a linker. Suitably, the linker isa protease-labile or a non-protease-labile linker. The linker issuitably a polypeptide and will be selected so as to allow binding ofthe polypeptides to their epitopes. If used for therapeutic purposes,the linker is suitably non-immunogenic in the subject to which thepolypeptides are administered. Suitably the polypeptides are allconnected by non-protease-labile linkers. Suitably the protease-labilelinker is of the format [-(G₄S)_(x)-BJB′-(G₄S)_(y)-]_(z) wherein J islysine or arginine, B is 0 to 5 amino acid residues selected from R, H,N, Q, S, T, Y, G, A, V, L, W, P, M, C, F, K or I, B′ is 0 to 5 aminoacid residues selected from R, H, N, Q, S, T, Y, G, A, V, L, W, M, C, F,K or I, x is 1 to 10; y is 1 to 10 and z is 1 to 10. Most suitably J islysine, B is 0 x is 1, y is 1 and z is 1. Suitably thenon-protease-labile linkers are of the format (G₄S)_(x). Most suitably xis 6.

Vectors and Hosts

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian and yeast vectors). Other vectors(e.g. non-episomal mammalian vectors) can be integrated into the genomeof a host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.replication defective retroviruses. adenoviruses and adeno-associatedviruses), which serve equivalent functions, and also bacteriophage andphagemid systems. The invention also relates to nucleotide sequencesthat encode polypeptide sequences or multivalent and/or multispecificconstructs. The term “recombinant host cell” (or simply “host cell”), asused herein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. Such terms are intended to refernot only to the particular subject cell but to the progeny of such acell.

In one aspect of the invention there is provided a vector comprising thepolynucleotide encoding the polypeptide or construct of the invention orcDNA comprising said polynucleotide. In a further aspect of theinvention there is provided a host cell transformed with said vector,which is capable of expressing the polypeptide or construct of theinvention. Suitably the host cell is a bacterium such as Escherchia colia yeast belonging to the genera Aspergillus, Saccharomyces,Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae orPichia pastoris.

Stability

Suitably, the polypeptide or construct of the present inventionsubstantially retains neutralisation ability and/or potency whendelivered orally and after exposure to the intestinal tract (forexample, after exposure to proteases of the small and/or large intestineand/or IBD inflammatory proteases). Such proteases includeenteropeptidase, trypsin, chymotrypsin, and irritable bowel diseaseinflammatory proteases (such as MMP3, MMP12 and cathepsin). Proteasesof, or produced in, the small and/or large intestine include proteasessourced from intestinal commensal microflora and/or pathogenic bacteria,for example wherein the proteases are cell membrane-attached proteases,excreted proteases and proteases released on cell lysis). Most suitablythe proteases are trypsin and chymotrypsin.

Suitably the intestinal tract is the intestinal tract of a dog, pig,human, cynomolgus monkey or mouse. The small intestine suitably consistsof the duodenum, jejunum and ileum. The large intestine suitablyconsists of the cecum, colon, rectum and anal canal. Suitably thepolypeptide or construct of the invention is substantially resistant toone or more proteases. The intestinal tract, as opposed to thegastrointestinal tract, consists of only the small intestine and thelarge intestine.

The polypeptide or construct of the present invention substantiallyretains neutralisation ability when suitably 10%, more suitably 20%,more suitably 30%, more suitably 40%, more suitably 50%, more suitably60%, more suitably 70%, more suitably 80%, more suitably 90%, moresuitably 95%, more suitably 100% of the original neutralisation abilityof the polypeptide of the invention or construct is retained afterexposure to proteases present in the small and/or large intestine and/orIBD inflammatory proteases.

Suitably the polypeptide or construct of the invention substantiallyretains neutralisation ability after exposure to proteases present inthe small and/or large intestine and/or IBD inflammatory proteases for,for example, up to at least 2, more suitably up to at least 3, moresuitably up to at least 4, more suitably up to at least 5, more suitablyup to at least 5.5, more suitably up to at least 16, more suitably up toat least 21 or more suitably up to at least 22 hours at 37 degrees C.

Suitably 10% or more, more suitably 20% or more, more suitably 30% ormore, more suitably 40% or more, more suitably 50% or more, moresuitably 60% or more, more suitably 70% or more of the neutralisationability of the polypeptide or construct of the invention is retainedafter 16 hours of exposure to conditions of the intestinal tract, moresuitably the small or large intestine, more suitably human faecalextract.

Suitably 10% or more, more suitably 20% or more, more suitably 30% ormore, more suitably 40% or more, more suitably 50% or more, moresuitably 60% or more, more suitably 70% or more of the neutralisationability of the polypeptide or construct of the invention is retainedafter suitably 4, 6 or 16 hours of exposure to mouse small intestinalsupernatant.

Suitably 10% or more, more suitably 15% or more, more suitably 20% ormore, more suitably 25% or more, more suitably 30% or more, moresuitably 35% or more, more suitably 40% or more of the neutralisationability of the polypeptide or construct of the invention is retainedafter 16 hours of exposure to mouse small intestinal supernatant.

Suitably 50% or more, more suitably 60% or more, more suitably 65% ormore, more suitably 70% or more, more suitably 75% or more, moresuitably 80% or more, more suitably 85% or more, more suitably 85% ormore of the neutralisation ability of the polypeptide or construct ofthe invention is retained after 16 hours of exposure to mouse smallintestinal supernatant.

Suitably 10% or more, more suitably 20% or more, more suitably 30% ormore, more suitably 40% or more, more suitably 50% or more, moresuitably 60% or more, more suitably 70% or more of the administered doseof polypeptides or constructs of the invention retain neutralisationability against TNF-alpha and remain in the faeces of a mouse,cynomolgus monkey and/or human (suitably excreted faeces or faecesremoved from the intestinal tract) after 1, 2, 3, 4, 5, 6 or 7 hours ofexposure to conditions of the intestinal tract.

A polypeptide of the invention or construct of the present inventionremains substantially intact when suitably 10%, more suitably 20%, moresuitably 30%, more suitably 40%, more suitably 50%, more suitably 60%,more suitably 70%, more suitably 80%, more suitably 90%, more suitably95%, more suitably 99%, most suitably 100% of the administered quantityof polypeptide of the invention or construct remains intact afterexposure to proteases present in the small and/or large intestine and/orIBD inflammatory proteases.

Suitably, the polypeptide of the invention or construct of the presentinvention remains substantially intact after exposure to the stomach,duodenum, jejunum or ileum of cynomolgus monkey for 5 hours. Suitably,the polypeptide of the invention or construct of the present inventionremains substantially intact after exposure to the caecum or colon ofcynomolgus monkey for 16 hours.

Suitably a polypeptide or construct of the present inventionsubstantially retains neutralisation ability when suitably 10%, moresuitably 20%, more suitably 30%, more suitably 40%, more suitably 50%,more suitably 60%, more suitably 70%, more suitably 80%, more suitably90%, more suitably 95%, more suitably 100% of the originalneutralisation ability of the polypeptide or construct is retained afterfreezing and thawing.

Therapeutic Use and Delivery

A therapeutically effective amount of a polypeptide, pharmaceuticalcomposition or construct of the invention, is an amount which iseffective, upon single or multiple dose administration to a subject, inneutralising TNF-alpha to a significant extent in a subject. Atherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the polypeptide, pharmaceutical composition or construct toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of thepolypeptide of the invention, pharmaceutical composition or constructare outweighed by the therapeutically beneficial effects. Thepolypeptide or construct of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.The polypeptide or construct of the invention can be in the form of apharmaceutically acceptable salt.

A pharmaceutical composition of the invention may suitably be formulatedfor oral, intramuscular, subcutaneous or intravenous delivery. Thepharmaceutical compositions of the invention may be in a variety offorms. These include, for example, liquid, semi-solid and solid dosageforms, such as liquid solutions (e.g., injectable and infusiblesolutions), dispersions or suspensions, tablets, pills, powders,liposomes and suppositories. Solid dosage forms are preferred. Thepolypeptide of the invention, pharmaceutical composition or constructmay be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like.

Typically, the pharmaceutical composition comprises a polypeptide orconstruct of the invention and a pharmaceutically acceptable diluent orcarrier. Examples of pharmaceutically acceptable carriers include one ormore of water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of thepolypeptide or construct of the invention. Pharmaceutical compositionsmay include antiadherents, binders, coatings, disintegrants, flavours,colours, lubricants, sorbents, preservatives, sweeteners, freeze dryexcipients (including lyoprotectants) or compression aids.

Most suitably, the polypeptide of the invention, pharmaceuticalcomposition or construct of the invention is administered orally. A keyproblem with oral delivery is ensuring that sufficient polypeptide,pharmaceutical composition or construct reaches the area of theintestinal tract where it is required. Factors which prevent apolypeptide, pharmaceutical composition or construct of the inventionreaching the area of the intestinal tract where it is required includethe presence of proteases in digestive secretions which may degrade apolypeptide, pharmaceutical composition or construct of the invention.Suitably, the polypeptide, pharmaceutical composition or construct ofthe invention are substantially stable in the presence of one or more ofsuch proteases by virtue of the inherent properties of the polypeptideor construct itself. Suitably, the polypeptide or construct of theinvention is lyophilised before being incorporated into a pharmaceuticalcomposition.

A polypeptide of the invention may also be provided with an entericcoating. An enteric coating is a polymer barrier applied on oralmedication which helps to protect the polypeptide from the low pH of thestomach. Materials used for enteric coatings include fatty acids, waxes,shellac, plastics, and plant fibers. Suitable enteric coating componentsinclude methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate (hypromellose acetate succinate),polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acidcopolymers, sodium alginate and stearic acid. Suitable enteric coatingsinclude pH-dependent release polymers.

These are polymers which are insoluble at the highly acidic pH found inthe stomach, but which dissolve rapidly at a less acidic pH. Thus,suitably, the enteric coating will not dissolve in the acidic juices ofthe stomach (pH ˜3), but will do so in the higher pH environment presentin the small intestine (pH above 6) or in the colon (pH above 7.0). ThepH-dependent release polymer is selected such that the polypeptide orconstruct of the invention will be released at about the time that thedosage reaches the small intestine.

A polypeptide, construct or pharmaceutical composition of the inventioncan be formulated into preparations for injection by dissolving,suspending or emulsifying them in an aqueous or non-aqueous solvent,such as vegetable or other similar oils, synthetic aliphatic acidglycerides, esters of higher aliphatic acids or propylene glycol; and ifdesired, with conventional additives such as solubilisers, isotonicagents, suspending agents, emulsifying agents, stabilisers andpreservatives. Acceptable carriers, excipients and/or stabilisers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid, glutathione, cysteine, methionineand citric acid; preservatives (such as ethanol, benzyl alcohol, phenol,m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkoniumchloride, or combinations thereof); amino acids such as arginine,glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid,isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan,methionine, serine, proline and combinations thereof; monosaccharides,disaccharides and other carbohydrates; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as gelatin or serumalbumin; chelating agents such as EDTA; sugars such as trehalose,sucrose, lactose, glucose, mannose, maltose, galactose, fructose,sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, andneuraminic acid; and/or non-ionic surfactants such as polysorbates, POEethers, poloxamers, Triton-X, or polyethylene glycol.

A pharmaceutical composition of the invention may be delivered topicallyto the skin (for example for use in the treatment of an autoimmunedisease such as psoriasis or eczema). Such a pharmaceutical compositionmay suitably be in the form of a cream, ointment, lotion, gel, foam,transdermal patch, powder, paste or tincture and may suitably includevitamin D3 analogues (e.g calcipotriol and maxacalcitol), steroids (e.g.fluticasone propionate, betamethasone valerate and clobetasolpropionate), retinoids (e.g. tazarotene), coal tar and dithranol.Topical medicaments are often used in combination with each other (e.g.a vitamin D3 and a steroid) or with further agents such as salicylicacid. If the pharmaceutical composition of the invention is to bedelivered topically for the treatment of psoriasis or eczema, suitably afurther substance considered to be effective in treating psoriasis oreczema may be included in the composition such as steroids especiallyClass 4 or Class 5 steroids such as hydrocortisone (eg 1% hydrocortisonecream); cyclosporin or similar macrolide agent or retinoids.

For all modes of delivery, the polypeptide, pharmaceutical compositionor construct of the invention may be formulated in a buffer, in order tostabilise the pH of the composition, at a concentration between 5-50, ormore suitably 15-40 or more suitably 25-30 g/litre. Examples of suitablebuffer components include physiological salts such as sodium citrateand/or citric acid. Suitably buffers contain 100-200, more suitably125-175 mM physiological salts such as sodium chloride. Suitably thebuffer is selected to have a pKa close to the pH of the composition orthe physiological pH of the patient.

Exemplary polypeptide or construct concentrations in a pharmaceuticalcomposition may range from about 1 mg/mL to about 200 mg/ml or fromabout 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200mg/mL.

An aqueous formulation of the polypeptide, construct or pharmaceuticalcomposition of the invention may be prepared in a pH-buffered solution,e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 toabout 6.0, or alternatively about 5.5. Examples of suitable buffersinclude phosphate-, histidine-, citrate-, succinate-, acetate-buffersand other organic acid buffers. The buffer concentration can be fromabout 1 mM to about 100 mM, or from about 5 mM to about 50 mM,depending, for example, on the buffer and the desired tonicity of theformulation.

The tonicity of the pharmaceutical composition may be altered byincluding a tonicity modifier. Such tonicity modifiers can be charged oruncharged chemical species. Typical uncharged tonicity modifiers includesugars or sugar alcohols or other polyols, preferably trehalose,sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol orlactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol).Typical charged tonicity modifiers include salts such as a combinationof sodium, potassium or calcium ions, with chloride, sulfate, carbonate,sulfite, nitrate, lactate, succinate, acetate or maleate ions(especially sodium chloride or sodium sulphate); or amino acids such asarginine or histidine. Suitably, the aqueous formulation is isotonic,although hypertonic or hypotonic solutions may be suitable. The term“isotonic” denotes a solution having the same tonicity as some othersolution with which it is compared, such as physiological salt solutionor serum. Tonicity agents may be used in an amount of about 5 mM toabout 350 mM, e.g., in an amount of 1 mM to 500 nM. Suitably, at leastone isotonic agent is included in the composition.

A surfactant may also be added to the pharmaceutical composition toreduce aggregation of the formulated polypeptide or construct and/orminimize the formation of particulates in the formulation and/or reduceadsorption. Exemplary surfactants include polyoxyethylensorbitan fattyacid esters (Tween), polyoxyethylene alkyl ethers (Brij),alkylphenylpolyoxyethylene ethers (Triton-X),polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), andsodium dodecyl sulfate (SDS). Examples of suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, andpolysorbate 80. Exemplary concentrations of surfactant may range fromabout 0.001% to about 10% w/v.

A lyoprotectant may also be added in order to protect the polypeptide orconstruct of the invention against destabilizing conditions during thelyophilization process. For example, known lyoprotectants include sugars(including glucose, sucrose, mannose and trehalose); polyols (includingmannitol, sorbitol and glycerol); and amino acids (including alanine,glycine and glutamic acid). Lyoprotectants can be included in an amountof about 10 mM to 500 mM.

The dosage ranges for administration of the polypeptide of theinvention, pharmaceutical composition or construct of the invention arethose to produce the desired therapeutic effect. The dosage rangerequired depends on the precise nature of the polypeptide of theinvention, pharmaceutical composition or construct, the route ofadministration, the nature of the formulation, the age of the patient,the nature, extent or severity of the patient's condition,contraindications, if any, and the judgement of the attending physician.Variations in these dosage levels can be adjusted using standardempirical routines for optimisation.

Suitable daily dosages of polypeptide of the invention, pharmaceuticalcomposition or construct of the invention are in the range of 50 ng-50mg per kg, such as 50 ug-40 mg per kg, such as 5-30 mg per kg of bodyweight. The unit dosage can vary from less than 100 mg, but typicallywill be in the region of 250-2000 mg per dose, which may be administereddaily or more frequently, for example 2, 3 or 4 times per day or lessfrequently for example every other day or once per week, once perfortnight or once per month.

In one aspect of the invention there is provided the use of thepolypeptide, pharmaceutical composition or construct of the invention inthe manufacture of a medicament for the treatment of autoimmune disease.In a further aspect of the invention there is provided a method oftreating autoimmune disease comprising administering to a person in needthereof a therapeutically effective amount of the polypeptide,pharmaceutical composition or construct of the invention.

The word ‘treatment’ is intended to embrace prophylaxis as well astherapeutic treatment. Treatment of diseases also embraces treatment ofexacerbations thereof and also embraces treatment of patients inremission from disease symptoms to prevent relapse of disease symptoms.

Combination Therapy

A pharmaceutical composition of the invention may also comprise one ormore active agents (e.g. active agents suitable for treating thediseases mentioned herein). It is within the scope of the invention touse the pharmaceutical composition of the invention in therapeuticmethods for the treatment of autoimmune diseases as an adjunct to, or inconjunction with, other established therapies normally used in thetreatment of autoimmune diseases.

For the treatment of IBD (such as Crohn's disease or ulcerativecolitis), possible combinations include combinations with, for example,one or more active agents selected from the list comprising:5-aminosalicylic acid, or a prodrug thereof (such as sulfasalazine,olsalazine or bisalazide); corticosteroids (e.g. prednisolone,methylprednisolone, or budesonide); immunosuppressants (e.g.cyclosporin, tacrolimus, methotrexate, azathioprine or6-mercaptopurine); anti-TNF-alpha antibodies (e.g., infliximab,adalimumab, certolizumab pegol or golimumab); anti-IL12/1L23 antibodies(e.g., ustekinumab); anti-IL6R antibodies or small molecule IL12/1L23inhibitors (e.g., apilimod); Anti-alpha-4-beta-7 antibodies (e.g.,vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies againstthe cell adhesion molecule alpha-4-integrin (e.g., natalizumab);antibodies against the 1L2 receptor alpha subunit (e.g., daclizumab orbasiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Sykinhibitors and prodrugs thereof (e.g., fostamatinib and R-406);Phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics;Dersalazine; semapimod/CPSI-2364; and protein kinase C inhibitors (e.g.AEB-071). The most suitable combination agents are infliximab,adalimumab, certolizumab pegol or golimumab.

Hence another aspect of the invention provides a pharmaceuticalcomposition of the invention in combination with one or more furtheractive agents, for example one or more active agents described above.

In a further aspect of the invention, the polypeptide, pharmaceuticalcomposition or construct is administered sequentially, simultaneously orseparately with at least one active agent selected from the list above.

Similarly, another aspect of the invention provides a combinationproduct comprising:

(A) a polypeptide, pharmaceutical composition or construct of thepresent invention; and

(B) one or more other active agents,

wherein each of components (A) and (B) is formulated in admixture with apharmaceutically-acceptable adjuvant, diluent or carrier. In this aspectof the invention, the combination product may be either a single(combination) formulation or a kit-of-parts. Thus, this aspect of theinvention encompasses a combination formulation including a polypeptide,pharmaceutical composition or construct of the present invention andanother therapeutic agent, in admixture with a pharmaceuticallyacceptable adjuvant, diluent or carrier.

The invention also encompasses a kit of parts comprising components:

(i) a polypeptide, pharmaceutical composition or construct of thepresent invention in admixture with a pharmaceutically acceptableadjuvant, diluent or carrier; and

(ii) a formulation including one or more other active agents, inadmixture with a pharmaceutically-acceptable adjuvant, diluent orcarrier, which components (i) and (ii) are each provided in a form thatis suitable for administration in conjunction with the other.

Component (i) of the kit of parts is thus component (A) above inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier. Similarly, component (ii) is component (B) above in admixturewith a pharmaceutically acceptable adjuvant, diluent or carrier. The oneor more other active agents (i.e. component (B) above) may be, forexample, any of the agents mentioned above in connection with thetreatment of autoimmune diseases such as IBD (e.g. Crohn's diseaseand/or ulcerative colitis). If component (B) is more than one furtheractive agent, these further active agents can be formulated with eachother or formulated with component (A) or they may be formulatedseparately. In one embodiment component (B) is one other therapeuticagent. In another embodiment component (B) is two other therapeuticagents. The combination product (either a combined preparation orkit-of-parts) of this aspect of the invention may be used in thetreatment or prevention of an autoimmune disease (e.g. the autoimmunediseases mentioned herein).

Suitably the polypeptide, pharmaceutical composition or construct of theinvention is for use as a medicament and more suitably for use in thetreatment of an autoimmune and/or inflammatory disease.

Autoimmune Diseases and/or Inflammatory Diseases

Autoimmune diseases develop when the immune system responds adversely tonormal body tissues. Autoimmune disorders may result in damage to bodytissues, abnormal organ growth and/or changes in organ function. Thedisorder may affect only one organ or tissue type or may affect multipleorgans and tissues. Organs and tissues commonly affected by autoimmunedisorders include blood components such as red blood cells, bloodvessels, connective tissues, endocrine glands such as the thyroid orpancreas, muscles, joints and skin An inflammatory disease is a diseasecharacterised by inflammation. Many inflammatory diseases are autoimmunediseases and vice-versa.

Autoimmune Diseases and/or Inflammatory Diseases of the GIT

The chronic inflammatory bowel diseases (IBD) Crohn's disease andulcerative colitis, which afflict both children and adults, are examplesof autoimmune and inflammatory diseases of the GIT (Hendrickson et al2002 Clin Microbiol Rev 15(1):79-94, herein incorporated by reference inits entirety). Ulcerative colitis is defined as a condition where theinflammatory response and morphologic changes remain confined to thecolon. The rectum is involved in 95% of patients. Inflammation islargely limited to the mucosa and consists of continuous involvement ofvariable severity with ulceration, edema, and hemorrhage along thelength of the colon (Hendrickson et al 2002 Clin. Microbiol Rev15(1):79-94, herein incorporated by reference in its entirety).Ulcerative colitis is usually manifested by the presence of blood andmucus mixed with stool, along with lower abdominal cramping which ismost severe during the passage of bowel movements. Clinically, thepresence of diarrhoea with blood and mucus differentiates ulcerativecolitis from irritable bowel syndrome, in which blood is absent. Unlikeulcerative colitis, the presentation of Crohn's disease is usuallysubtle, which leads to a later diagnosis. Factors such as the location,extent, and severity of involvement determine the extent ofgastrointestinal symptoms. Patients who have ileocolonic involvementusually have postprandial abdominal pain, with tenderness in the rightlower quadrant and an occasional inflammatory mass. Symptoms associatedwith gastroduodenal Crohn's disease include early satiety, nausea,emesis, epigastric pain, or dysphagia. Perianal disease is common, alongwith anal tags, deep anal fissures, and fistulae (Hendrickson et al 2002Clin Microbiol Rev 15(1):79-94, herein incorporated by reference in itsentirety).

In these diseases the TNF-alpha is produced in the lamina propriaunderlying the gastrointestinal epithelium. This epithelium is disruptedin IBD and facilitates transport of the immunoglobulin chain variabledomain into the lamina propria—the site of TNF-alpha production andbiological action in these diseases (see Example 8). Other diseases ofthe GIT include for example the inflammatory disease mucositis (suitablydrug- and radiation induced-mucositis) where inflammatory lesions arepresent in the mucosa disrupting the epithelial tight junctions whichalso allow the immunoglobulin chain variable domain access to the siteof TN F-alpha production. In mucositis the lesions can occur anywherefrom mouth to anus and for mouth and oesophagus lesions a mouthwash orcream preparation containing the variable domain may be used. For analand rectal lesions, suppositories, creams or foams containing thevariable domain would be suitable for topical application. Theimmunoglobulin chain variable domains will be cleared from the laminapropria or other inflammatory sites via absorption into the bloodstreamat sites of inflammation or via lympatic clearance and subsequent entryinto the bloodstream. The domains will therefore reach the liver via thebloodstream and will be cleared via glomerular filtration in the kidney.There is therefore good rationale that the domains will functiontherapeutically in diseases such as autoimmune hepatitis, type IIdiabetes and glomerular nephritis.

Suitably the polypeptide, pharmaceutical composition or construct of theinvention is used in the treatment of an autoimmune and/or inflammatorydisease of the GI (gastrointestinal) tract where TNF-alpha contributesto the pathology of such disease.

Suitably the polypeptide, pharmaceutical composition or construct of theinvention is for use in the treatment of an autoimmune and/orinflammatory disease of the GI tract selected from the list consistingof Crohn's disease, ulcerative colitis, irritable bowel disease,diabetes type II, glomerulonephritis, autoimmune hepatitis, Sjogren'ssyndrome, celiac disease and drug- or radiation-induced mucositis (mostsuitably Crohn's disease).

Oral delivery of the immunoglobulin chain variable domain will ideallytreat inflammatory diseases where TNF-alpha contributes to at least aproportion of the pathology and the immunoglobulin chain variable domaincan access the tissue where the TNF-alpha is biologically active.

Autoimmune Diseases and/or Inflammatory Diseases of the Skin

Psoriasis is a debilitating autoimmune, dermatological, disease. Plaquepsoriasis, the most common form of the disease, is characterized by redskin covered with silvery scales. Histologically the picture is one ofdisordered differentiation and hyperproliferation of keratinocyteswithin the psoriatic plaque with inflammatory cell infiltrates (Ortonne,1999 Brit J Dermatol 140(suppl 54):1-7). The psoriatic skin lesions areinflammatory, red, sharply delimited plaques of various shapes withcharacteristic silvery lustrous scaling. The term psoriasis includespsoriasis and the symptoms of psoriasis including erythema, skinthickening/elevation and scaling.

Biological agents of use in the treatment of psoriasis include anti-TNF-alpha therapies (such as monoclonal antibodies against TNF, e.g.adalimumab and infliximab, or TNF-alpha receptor fusion proteins such asetanercept), humanised antibodies to CD11a (efalizumab) or agents whichbind to CD2 such as alefacept (thereby blocking the CD2 LFA3interaction). It should be noted that not all of the biological agentslisted here have been approved for use in the treatment of psoriasis.

The polypeptide of the invention may be incorporated into acream/ointment or other topical carrier for administration toinflammatory skin lesions where TNF-alpha contributes to the pathologyof such lesions.

Suitably the polypeptide, pharmaceutical composition or construct of theinvention is for use in the treatment of an autoimmune and/orinflammatory disease of the skin selected from the list consisting ofpemphigus, psoriasis, eczema and scleroderma.

Suitably the polypeptide, pharmaceutical composition or construct is foruse in the treatment of other autoimmune/inflammatory diseases in whichTNF-alpha is responsible for a proportion of the pathology observed.

Preparative Methods

Polypeptides of the invention can be obtained and manipulated using thetechniques disclosed for example in Green and Sambrook 2012 MolecularCloning: A Laboratory Manual 4th Edition Cold Spring Harbour LaboratoryPress.

Monoclonal antibodies can be produced using hybridoma technology, byfusing a specific antibody-producing B cell with a myeloma (B cellcancer) cell that is selected for its ability to grow in tissue cultureand for an absence of antibody chain synthesis (Köhler and Milstein 1975Nature 256:495-497 and Nelson et al 2000 Molecular Pathology53(3):111-117, herein incorporated by reference in their entirety).

A monoclonal antibody directed against a determined antigen can, forexample, be obtained by:

a) immortalizing lymphocytes obtained from the peripheral blood of ananimal previously immunized with a determined antigen, with an immortalcell and preferably with myeloma cells, in order to form a hybridoma,

b) culturing the immortalized cells (hybridoma) formed and recoveringthe cells producing the antibodies having the desired specificity.

Alternatively, the use of a hybridoma cell is not required. Accordingly,monoclonal antibodies can be obtained by a process comprising the stepsof:

a) cloning into vectors, especially into phages and more particularlyfilamentous bacteriophages, DNA or cDNA sequences obtained fromlymphocytes especially peripheral blood lymphocytes of an animal(suitably previously immunized with determined antigens),

b) transforming prokaryotic cells with the above vectors in conditionsallowing the production of the antibodies,

c) selecting the antibodies by subjecting them to antigen-affinityselection, d) recovering the antibodies having the desired specificity.

Methods for immunizing camelids, cloning the VHH repertoire of B cellscirculating in blood (Chomezynnski and Sacchi 1987 Anal Biochem162:156-159), and isolation of antigen-specific VHHs from immune(Arbabi-Ghahroudi et al 1997 FEBS Lett 414:521-526) and nonimmune (Tanhaet al 2002 J Immunol Methods 263:97-109) libraries using phage, yeast,or ribosome display are known (WO92/01047, Nguyen et al 2001 Adv Immunol79:261-296 and Harmsen et al 2007 Appl Microbiol Biotechnol77(1):13-22). These references are herein incorporated by reference intheir entirety.

Antigen-binding fragments of antibodies such as the scFv and Fvfragments can be isolated and expressed in E. coli (Miethe et al 2013 JBiotech 163(2):105-111, Skerra et al 1988 Science 240(4855):1038-1041and Ward et al Nature 1989 341:544-546, herein incorporated by referencein their entirety).

Mutations can be made to the DNA or cDNA that encode polypeptides whichare silent as to the amino acid sequence of the polypeptide, but whichprovide preferred codons for translation in a particular host. Thepreferred codons for translation of a nucleic acid in, e.g., E. coli andS. cerevisiae, are known.

Mutation of polypeptides can be achieved for example by substitutions,additions or deletions to a nucleic acid encoding the polypeptide. Thesubstitutions, additions or deletions to a nucleic acid encoding thepolypeptide can be introduced by many methods, including for exampleerror-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis (Ling et al 1997 Anal Biochem254(2):157-178, herein incorporated by reference in its entirety), genereassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligationreassembly (SLR) or a combination of these methods. The modifications,additions or deletions to a nucleic acid can also be introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, or acombination thereof.

In particular, artificial gene synthesis may be used (Nambiar et al 1984Science 223:1299-1301, Sakamar and Khorana 1988 Nucl. Acids Res14:6361-6372, Wells et al 1985 Gene 34:315-323 and Grundstrom et al 1985Nucl. Acids Res 13:3305-3316, herein incorporated by reference in theirentirety). A gene encoding a polypeptide of the invention can besynthetically produced by, for example, solid-phase DNA synthesis.Entire genes may be synthesized de novo, without the need for precursortemplate DNA. To obtain the desired oligonucleotide, the building blocksare sequentially coupled to the growing oligonucleotide chain in theorder required by the sequence of the product. Upon the completion ofthe chain assembly, the product is released from the solid phase tosolution, deprotected, and collected. Products can be isolated byhigh-performance liquid chromatography (HPLC) to obtain the desiredoligonucleotides in high purity (Verma and Eckstein 1998 Annu RevBiochem 67:99-134).

Expression of immunoglobulin chain variable domains such as VHs and VHHscan be achieved using a suitable expression vector such as a prokaryoticcell such as bacteria, for example E. coli (for example according to theprotocols disclosed in WO94/04678, which is incorporated herein byreference and detailed further below). Expression of immunoglobulinchain variable domains such as VHs and VHHs can also be achieved usingeukaryotic cells, for example insect cells, CHO cells, Vero cells orsuitably yeast cells such as yeasts belonging to the genera Aspergillus,Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably S.cerevisiae is used (for example according to the protocols disclosed inWO94/025591, which is incorporated herein by reference and detailedfurther below).

Specifically, VHHs can be prepared according to the methods disclosed inWO94/04678 using E. coli cells by a process comprising the steps of:

a) cloning in a Bluescript vector (Agilent Technologies) a DNA or cDNAsequence coding for the VHH (for example obtained from lymphocytes ofcamelids or produced synthetically) optionally including a His-tag,

b) recovering the cloned fragment after amplification using a 5′ primerspecific for the VHH containing an XhoI site and a 3′ primer containingthe SpeI site having the sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG(SEQ ID NO: 58),

c) cloning the recovered fragment in phase in the Immuno PBS vector(Huse et al 1989 Science 246 (4935):1275-1281, herein incorporated byreference in its entirety) after digestion of the vector with XhoI andSpeI restriction enzymes,

d) transforming host cells, especially E. coli by transfection with therecombinant Immuno PBS vector of step c,

e) recovering the expression product of the VHH coding sequence, forinstance by affinity purification such as by chromatography on a columnusing Protein A, cation exchange, or a nickel-affinity resin if the VHHincludes a His-tag.

Alternatively, immunoglobulin chain variable domains such as VHs andVHHs are obtainable by a process comprising the steps of:

a) obtaining a DNA or cDNA sequence coding for a VHH, having adetermined specific antigen binding site,

b) amplifying the obtained DNA or cDNA, using a 5′ primer containing aninitiation codon and a HindIII site, and a 3′ primer containing atermination codon having a XhoI site,

c) recombining the amplified DNA or cDNA into the HindIII (position2650) and XhoI (position 4067) sites of a plasmid pMM984 (Merchlinsky etal 1983 J. Virol. 47:227-232, herein incorporated by reference in itsentirety),

d) transfecting permissive cells especially NB-E cells (Faisst et al1995 J Virol 69:4538-4543, herein incorporated by reference in itsentirety) with the recombinant plasmid,

e) recovering the obtained products.

Further, immunoglobulin chain variable domains such as VHHs or VHs canbe produced using E. coli or S. cerevisiae according to the methodsdisclosed in Frenken et al 2000 J Biotech 78:11-21 and WO99/23221(herein incorporated by reference in their entirety) as follows:

After taking a blood sample from an immunised llama and enriching thelymphocyte population via Ficoll (a neutral, highly branched, high-mass,hydrophilic polysaccharide which dissolves readily in aqueoussolutions—Pharmacia) discontinuous gradient centrifugation, isolatingtotal RNA by acid guanidium thiocyanate extraction (Chomezynnski andSacchi 1987 Anal Biochem 162:156-159), and first strand cDNA synthesis(e.g. using a cDNA kit such as RPN 1266 (Amersham)), DNA fragmentsencoding VHH and VH fragments and part of the short or long hinge regionare amplified by PCR using the specific primers detailed on pages 22 and23 of WO99/23221. Upon digestion of the PCR fragments with PstI andHindIII or BstEII, the DNA fragments with a length between about 300 and450 bp are purified via agarose gel electrophoresis and ligated in theE. coli phagemid vector pUR4536 or the episomal S. cerevisiae expressionvector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboomet al 1991 Nucl Acid Res 19:4133-4137, herein incorporated by referencein its entirety) and contains the lacl^(q) gene and unique restrictionsites to allow the cloning of the llama VHH and VH genes. pUR4548 isderived from pSY1 (Harmsen et al 1993 Gene 125:115-123, hereinincorporated by reference in its entirety). From this plasmid, theBstEII site in the leu2 gene is removed via PCR and the cloning sitesbetween the SUC2 signal sequence and the terminator are replaced inorder to facilitate the cloning of the VH/VHH gene fragments.

The VH/VHHs have the c-myc tag at the C-terminus for detection.Individual E. coli JM109 colonies are transferred to 96 well microtiterplates containing 150 ml 2TY medium supplemented with 1% glucose and 100mg L⁻¹ ampicillin. After overnight growth (37 degrees C.), the platesare duplicated in 2TY medium containing 100 mg L⁻¹ ampicillin and 0.1 mMIPTG. After another overnight incubation and optionally freezing andthawing, cells are centrifuged and pelleted and the supernatant can beused in an ELISA. Individual S. cerevisiae colonies are transferred totest tubes containing selective minimal medium (comprising 0.7% yeastnitrogen base, 2% glucose, supplemented with the essential amino acidsand bases) and are grown for 48 h at 30 degrees C. Subsequently, thecultures are diluted ten times in YPGal medium (comprising 1% yeastextract, 2% bacto peptone and 5% galactose). After 24 and 48 h ofgrowth, the cells are pelleted and the culture supernatant can beanalysed in an ELISA. Absorbance at 600 nm (0D600) is optionallymeasured.

Further, immunoglobulin chain variable domains such as VH/VHHs can beproduced using S. cerevisiae using the procedure as follows:

Isolate a naturally-occurring DNA sequence encoding the VH/VHH or obtaina synthetically produced DNA sequence encoding the VH/VHH, including a5′-UTR, signal sequence, stop codons and flanked with SacI and HindIIIsites (such a synthetic sequence can be produced as outlined above orfor example may be ordered from a commercial supplier such as Geneart(Life Technologies)).

Use the restriction sites for transfer of the VH/VHH gene to themulti-copy integration (MCI) vector pUR8569 or pUR8542, as follows. Cutthe DNA sequence encoding the VHH optionally contained within a shuttlevector, cassette or other synthetic gene construct and the MCI vectorwith SacI and HindIII using: 25 ul VHH DNA (Geneart plasmid or MCIvector), 1 ul SacI, 1 ul HindIII, 3 ul of a suitable buffer for doubledigestion such as NEB buffer 1 (New England Biolabs) overnight at 37degrees C. Run 25 ul of digested DNA encoding the VHH and 25 ul ofdigested MCI vector on a 1.5% agarose gel with 1×TAE buffer and thenperform gel extraction for example using QIAquick Gel Extraction Kit(Qiagen)). Set-up a ligation of digested MCI vector and digested DNAencoding the VH/VHH as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul10× ligase buffer, 1 ul T4 DNA ligase, and ddH₂O. Then perform ligationovernight at 16 degrees C.

Next transform the E. coli cells. For chemical competent XL-1 bluecells, thaw 200 ul heat competent XL-1 blue cells and add 5 ul ligationmix on ice for about 30 minutes followed by heat shock for 90 seconds at42 degrees C. Then add 800 ul Luria-Bertani low salt medium supplementedwith 2% glucose and recover cells for 2 hours at 37 degrees C. Platecells on Luria-Bertani agar and ampicillin (100 ug/ml) plates and keepovernight at 37 degrees C. For electro competent TG1 E. coli cells, usean electroporation cuvette. In the electroporation cuvette: thaw 50 ulelectro competent TG1 cells and 1 ul ligation mix on ice for about 15minutes. Place the cuvette in the holder and pulse. Add 500 ul of 2TYmedium and recover cells for 30 minutes at 37 degrees C. Plate 100 ul ofcells on Luria-Bertani, agar, containing ampicillin (100 ug/ml) and 2%glucose plates. Keep plates at 37 degrees C. overnight.

After cloning of the VH/VHH gene into E. coli as detailed above, S.cerevisiae can be transformed with the linearized MCI vector. Beforetransformation is carried out, some steps are performed: (i) the DNAshould be changed from circular to linear by digestion or else the DNAcannot be integrated into the yeast genome and (ii) the digested DNAshould be cleaned of impurities by ethanol precipitation. Also, duringthe transformation process, the yeast cells are made semi-permeable sothe DNA can pass the membrane.

Preparation for yeast transformation: perform a HpaI digestion of themidi-prep prepared from the selected E. coli colony expressing theVH/VHH gene as follows. Prepare a 100 ul solution containing 20 ng ofmidi-prep, 5 ul HpaI, 10 ul of appropriate buffer such as NEB4 buffer(BioLabs), and ddH₂O.

Cut the DNA with the HpaI at room temperature overnight. Next perform anethanol precipitation (and put to one side a 5 ul sample from HpaIdigestion). Add 300 ul ethanol 100% to 95 ul HpaI digested midiprep,vortex, and spin at full speed for 5 minutes. Carefully decant when apellet is present, add 100 ul of ethanol 70%, then spin again for 5minutes at full speed. Decant the sample again, and keep at 50-60degrees C. until the pellet is dry. Re-suspend the pellet in 50 ulddH₂O. Run 5 ul on a gel beside the 5 ul HpaI digested sample.

Yeast transformation: prepare YNBglu plates. Use 10 g agar+425 ml water(sterilised), 25 ml filtered 20×YNB (3.35 g YNB (yeast nitrogen base) in25 ml sterilized H₂O) and 50 ml sterile 20% glucose and pour into petridishes. Pick one yeast colony from the masterplate and grow in 3 ml YPD(Yeast Extract Peptone Dextrose) overnight at 30 degrees C. Next dayprepare about 600 ml YPD and use to fill 3 flasks with 275 ml, 225 mland 100 ml YPD. Add 27.5 ul yeast YPD culture to the first flask and mixgently. Take 75 ml from the first flask and put this in the secondflask, mix gently. Take 100 ml from the second flask and put in thethird one, mix gently. Grow until reaching an OD660 of between 1 and 2.Divide the flask reaching this OD over 4 Falcon tubes, ±45 ml in each.Spin for 2 minutes at 4200 rpm. Discard the supernatant. Dissolve thepellets in two Falcon tubes with 45 ml H₂O (reducing the number of tubesfrom 4 to 2). Spin for 2 minutes at 4200 rpm. Dissolve the pellets in 45ml H₂O (from 2 tubes to 1). Spin for 2 minutes at 4200 rpm. Gentlydissolve the pellets in 5 ml lithium acetate (LiAc) (100 mM), and spinfor a few seconds. Carefully discard some LiAc, but retain over half ofthe LiAc in the tube. Vortex the cells, boil carrier DNA for 5 minutesand quickly chill in ice-water. Add to a 15 ml tube containing: 240 ulPEG, 50 ul cells, 36uLiAc (1M), 25 ul carrier DNA, 45 ul ethanolprecipitated VH/VHH. Mix gently after each step (treat the blank samplethe same, only without ethanol precipitated VH/VHH). Incubate for 30minutes at 30 degrees C., gently invert the tube 3-4 times, then heatshock for 20-25 minutes at 42 degrees C. Spin up to 6000 rpm for a brieftime. Gently remove the supernatant and add 250 ul ddH₂O and mix. Streakall of it on an YNBglu plate until plates are dry and grow for 4-5 daysat 30 degrees C. Finally, prepare YNBglu plates by dividing plates in 6equal parts, number the parts 1 to 6, inoculate the biggest colony andstreak out number 1. Repeat for other colonies from big to small from 1to 6. Grow at 30 degrees C. for 3-4 days large until colonies areproduced. The VH/VHH clones are grown using glucose as a carbon source,and induction of VH/VHH expression is done by turning on theGalactose-7-promoter by adding 0.5% galactose. Perform a 3 mL smallscale culture to test the colonies and choose which one shows the bestexpression of the VH or VHH. This colony is then used in purification.

Purification: the VH/VHH is purified by cation exchange chromatorgraphywith a strong anion resin (such as Capto S). On day 1, inoculate theselected yeast colony expressing the VH/VHH in 5 ml YPD medium (YPmedium+2% glucose) and grow the cells in 25 mL sealed sterile tubes at30 degrees C. overnight (shaking at 180 rpm). On day 2, dilute the 5 mlovernight culture in 50 mL freshly prepared YP medium+2% glucose+0.5%galactose, grow the cells in 250 ml aerated baffled flasks at 30 degreesC. for two nights (shaking at 180 rpm). On day 4, spin the cells down ina centrifuge at 4200 rpm for 20 min. Cation exchange purification stepusing a strong anion resin: adjust the pH of the supernatant containingthe ligand to 3.5. Wash 0.75 ml resin (+/−0.5 mL slurry) per of 50 mLsupernatant with 50 mL of ddH₂O followed by three washes with bindingbuffer. Add the washed resin to the supernatant and incubate thesuspension at 4 degrees C. on a shaker for 1.5 hours. Pellet theresin-bound VH/VHH by centrifugation at 500 g for 2 minutes and wash itwith wash buffer. Decant supernatant and re-suspend the resin with 10 mLof binding buffer. Put a filter in a PD-10 column, pour the resin in thecolumn and let the resin settle for a while, then add a filter above theresin. Wait until all binding buffer has run through. Elute the VH/VHHwith 6×0.5 ml elution buffer. Collect the elution fractions in eppendorftubes. Measure the protein concentration of the 6 eluted fractions witha Nanodrop. Pool the fractions that contain the VHH and transfer thesolution into a 3,500 Da cutoff dialysis membrane. Dialyze the purifiedprotein solution against 3 L of PBS overnight at 4 degrees C. On day 5,dialyze the purified protein solution against 2 L of fresh PBS for anadditional 2 hours at 4 degrees C. Finally, calculate the finalconcentration by BCA.

Although discussed in the context of the VH/VHH, the techniquesdescribed above could also be used for scFv, Fab, Fv and other antibodyfragments if required.

Multiple antigen-binding fragments (suitably VH/VHHs) can be fused bychemical cross-linking by reacting amino acid residues with an organicderivatising agent such as described by Blattler et al Biochemistry24:1517-1524 (herein incorporated by reference in its entirety).Alternatively, the antigen-binding fragments may be fused genetically atthe DNA level i.e. a polynucleotide construct formed which encodes thecomplete polypeptide construct comprising one or more antigen-bindingfragments. One way of joining multiple antigen-binding fragments via thegenetic route is by linking the antigen-binding fragment codingsequences either directly or via a peptide linker. For example, thecarboxy-terminal end of the first antigen-binding fragment may be linkedto the amino-terminal end of the next antigen-binding fragment. Thislinking mode can be extended in order to link antigen-binding fragmentsfor the construction of tri-, tetra-, etc. functional constructs. Amethod for producing multivalent (such as bivalent) VHH polypeptideconstructs is disclosed in WO 96/34103 (herein incorporated by referencein its entirety).

Suitably, the polypeptide of the invention (in particular, a VHH of theinvention) can be produced in a fungus such as a yeast (for example, S.cerevisiae) comprising growth of the fungus on a medium comprising acarbon source wherein 50-100 wt % of said carbon source is ethanol,according to the methods disclosed in WO02/48382. Large scale productionof VHH fragments in S. cerevisiae is described in Thomassen et al 2002Enzyme and Micro Tech 30:273-278 (herein incorporated by reference inits entirety).

In one aspect of the invention there is provided a process for thepreparation of the polypeptide or construct of the invention comprisingthe following steps:

i) cloning into a vector, such as a plasmid, the polynucleotide of theinvention,

ii) transforming a cell, such as a bacterial cell or a yeast cellcapable of producing the polypeptide or construct of the invention, withsaid vector in conditions allowing the production of the polypeptide orconstruct,

iii) recovering the polypeptide or construct, such as by affinitychromatography.

The present invention will now be further described by means of thefollowing non-limiting examples.

EXAMPLES Example 1: Llama Immunisation, Phage Display, ImmunoglobulinChain Variable Domain Selection and Propagation

1.1 Immunisation Protocol 1

Initially, two llamas (llama 33 and llama 35) each received 3 injections(on days 0, 14 and 28) of soluble human recombinant TNF-alpha (100 uginjected intra-muscularly, after mixing 1:1 with Stimune adjuvant)followed by 2 injections (on days 56 and 70) with THP-1 cells that hadbeen pre-activated by incubation with bacterial lipopolysaccharide toincrease membrane TNF-alpha expression (10⁷ THP-1 cells injectedsubcutaneously in 1 ml PBS). A final boosting immunisation with bothsoluble TNF-alpha and activated THP-1 cells was given on day 84 andblood was drawn 9 days later for the isolation of peripheral bloodlymphocytes and extraction of RNA for library construction.

1.2 Immunisation Protocol 2

After resting for a period of four months the llamas were re-immunisedwith two injections a week apart of CHO Flp-In cells that had beenengineered to express a non-cleavable trans-membrane form of humanTNF-alpha. Blood was drawn two weeks later for the isolation ofperipheral blood lymphocytes and RNA extracted for library construction.Serum immune responses to immunisations were assessed for each of thellamas at several time-points by measuring the binding of heavy chainonly antibodies to immobilised human TNF-alpha using a plate ELISAformat and detection with rabbit polyclonal anti-variable domain serumand donkey anti-rabbit IgG coupled to horseradish peroxidase (HRP).Titration curves obtained showed that each of the llamas had respondedto the first round of immunisations giving high titres ofTNF-alpha-binding IgG heavy chain antibodies. Raised serum antibodytitres were also noted following the second round of immunisationsalthough these were somewhat lower for both animals.

1.3 Phage Library Construction

Blood cells collected from each llama at the end of each immunisationphase were used to generate four separate phage display libraries (#33,#35, #33NEW and #35NEW). Total RNA was extracted from the peripheralblood lymphocytes that were isolated from each of the immunised llamas(llamas 33 and 35). The RNA was then used to generate cDNA using reversetranscriptase and primers or random octameric oligonucleotides. PCR wasthen performed to amplify specifically the variable domain region ofheavy chain only antibodies, using suitable primers. cDNA fragmentsencoding the variable domain repertoire were cloned into a phagemidvector and the library introduced into E. coli. Phages were rescued fromthe bacteria containing the libraries by inoculating respectively 40 and51 ul from the glycerol stock in 50 ml medium containing glucose andampicillin. When cultures were at log-phase, helper phage was added toinfect the cultures and produce phages. Next day, produced phages wereprecipitated from the culture supernatant using a PEG/NaCl solution. Thenumber of phages was determined by titration of the solution andinfecting log-phase E. coli TG1 with the different phage dilutions. Thehighest dilution still giving rise to formation of colonies was used tocalculate the phage titers. For selection on sTNF-alpha, a sterile96-well (such as Maxisorp) plate was coated overnight at 4 degrees C.with either 1000, 250 or 63 ng/well or PBS only. Next day the wells ofthe plate were blocked with 4% Marvel in PBS, then the precipitatedphages, which were preincubated with 2% marvel/PBS, were added to thewells. After extensive washing with PBS-Tween and PBS, bound phages wereeluted either using alkaline pH shock (0.1 M Triethylamine) for 15minutes and neutralized with 1 M Tris-HCl pH7.5 (total elution), or withcompetitive elution, using a 10 times excess of the TNF receptor, fortwo hours. The number of eluted phages was determined by titration ofthe elutions from the different wells followed by infection of log-phaseE. coli TG1. About half of the eluted phages were rescued by infectinglog-phase TG1 and selecting in medium containing ampicillin and glucose.

1.4 Library Selections for Phage with Human and Cynomolgous MonkeyTNF-Alpha Binding Activity

Variable domains with TNF-alpha antagonistic activity were isolated fromthe four phage display libraries by binding of the phage to humanTNF-alpha followed by elution of TNF-alpha-specific variable domainswith an excess of soluble TNFR2-Fc fusion protein (etanercept), or bybatch elution. A second round of selection was then performed by bindingof the eluted human TNF-alpha specific phage to cynomolgus monkeyTNF-alpha such that a pool of variable domains with cross-species TNF-alpha-binding activity could be obtained.

1.4.1 First Round Library Selections of Phage Displaying VariableDomains with Human TNF-Alpha Binding Activity

Phage libraries were derived from llama 33 and llama 35 in respect ofeach stage of immunisation (first stage immunisation: Library #33 andLibrary #35; second stage immunisation: Library #33NEW and Library#35NEW). A first round of selection was performed to isolate phagedisplaying variable domains with TNF-alpha-binding activity by panningon human TNF-alpha.

Soluble recombinant human TNF-alpha (1000, 250 or 63 ng/well) was coateddirectly onto wells of a hydrophilised polypropylene microwell (HPM)plate, (for example, Nunc maxisorp) and phages were allowed to attach.After extensive washing, bound phages were collected using either (i)non-selective elution by alkaline pH shock or (ii) by selectivedisplacement of TNF-alpha-bound phages by the addition of TNFR2-Fc(etanercept 100, 25 and 6.3 ug/ml, 2h).

Library #33 and Library #35—The numbers of phages eluted from controlwells (no TNF-alpha and PBS control) were low and for both librariesthere were concentration-related enrichments of phages eluted from thewells that had been pre-coated with TNF-alpha, using either of theelution methods. Overall, the enrichments of TNF-binding phage achievedin the first round selections were about 100-fold for both Libraries #33and #35.

Library #33NEW and Library #35NEW—The number of clones recovered fromthe first rounds of selection of the libraries prepared from there-immunised llamas were found to be low. This may have reflected alower immune response of the llamas to the mTNF-alpha-CHO cells (lowernumber of TNF-alpha-reactive clones in total) or that the response ledto the generation of mTNF-alpha reactive clones but that only a limitednumber of these cross-reacted with the soluble form of TNF-alpha. Phageseluted in the first round selections with TNFR2-Fc (etanercept) from thewells coated with 1000 ng/ml human TNF-alpha were expanded yieldingsuspensions with titres of approximately 2×10¹² phages/ml.

1.4.2 Second Round Selections for Phage Displaying Immunoglobulin ChainVariable Domains with Human and Cynomolgus Monkey TN F-Alpha BindingActivity

Library #33 and Library #35—Second round selections were performed byattachment of the selected phages to soluble human or cynomolgus monkeyTNF-alpha (500, 125 or 31 ng/well or PBS) that had been pre-adsorbedonto the well surface of a HPM plate. Phages bound to human TNF-alphawere eluted with soluble TNFR2-Fc (50, 12.5 and 3.1 ug/ml respectively)while those bound to cynomolgus monkey TNF-alpha were eluted usingalkaline pH shock. Recoveries from the selections on cynomolgus monkeyTNF-alpha were lower than those on human TNF-alpha, possibly due tolower frequencies in both libraries of phages expressing variabledomains that are cross-reactive with both human and cynomolgus monkeyTNF-alpha.

Library #33NEW and Library #35NEW—Second round selections were performedby attachment of the selected phages to soluble cynomolgus monkeyTNF-alpha (1000, 250 or 63 ng/well or PBS) that had been pre-adsorbedonto the well surface of a HPM plate. Bound phages were eluted usingalkaline pH shock.

Example 2: Primary Evaluation of Periplasmic Supernatants from RandomlySelected Immunoglobulin Chain Variable Domain Clones

2.1 Propagation and Generation of Periplasmic Extracts for PrimaryEvaluation

Phage present in eluates from the first round selections on humanTNF-alpha and second round selections on cynomolgus monkey TNF-alphawere used to infect E. coli TG1 cells. Colonies were randomly picked(186 clones from Libraries #33 and #35 into master plates M60-M63; 96clones from Libraries #33NEW and Library #35NEW into master plate M65)and propagated to generate periplasmic supernatants by sequentialcentrifugation, resuspension in 1×PBS, freeze-thaw fracture andcentrifugation of the propagated cultures (M60-63 and M65 are alsoreferred to as ‘MP60-63’ and ‘MP65’, respectively; periplasmicsupernatants are also termed periplasmic fractions (PF) or extracts).

The periplasmic extracts were screened to test for their ability to (i)inhibit the binding of soluble human TNF-alpha to TNFR2-Fc in a plateELISA and (ii) neutralise TNF-alpha-induced cytotoxicity in the fixedconcentration L929 assay. In the L929 assay the cytotoxic effect ofTNF-alpha (0.5 ng/ml) is considered to be mediated via TNFR1, sovariable domains with inhibitory activities in bothTNF-alpha-TNFR2-binding and L929 assays are expected to suppressTNF-alpha-responses mediated via either receptor.

2.2.1 Evaluating TNF-Alpha Binding and Neutralising Activity (MasterPlates M60-M63)

L929 cytotoxicity and ELISA assays were performed on all 186 periplasmicfractions obtained from clones selected from Library #33 and Library#35. The clones were arranged on 4 master plates M60, M61, M62 and M63and periplasmic fractions were produced from these master plates. M60-61contained clones randomly picked from Library #33 and #35. M62-63contained clones selected from the same libraries after 2 subsequentsteps of selection were performed as follows: (i) 10 ug/mL hsTN F-alphaand etanercept for the elution; (ii) hsTNF-alpha (5 ug/mL and 0.3 ug/mL)or cynomolgus sTNF-alpha (5 ug/mL and 0.3 ug/mL) followed by eitherselective elution with etanercept or complete elution withtriethylamine. All the 186 PF were analysed by concentration-independentL929 cytotoxicity assay using resazurin.

2.2.2 Screening of Periplasmic Supernatants by ELISA (Master PlatesM60-63)

The TNF-alpha concentration used for screening was 1.25 ng/ml.Periplasmic supernatants were analysed at 1/20 dilution (M60 and M61) or1/10 dilution (M62 and M63) in 1% BSA. TNF-alpha and periplasmicsupernatants were made up at twice the assay concentration, and thenmixed together 1:1. Triplicate determinations were carried out for eachsupernatant. In addition, adalimumab at 10 nM was used as a positiveneutralising antibody control. On each ELISA plate, a limited TNF-alphadose-response (0-5 ng/ml) was also incorporated. ELISA plates foranalyses of the periplasmic supernatants were coated overnight with 0.7ug/ml, 50 ul/well etanercept. After washing and blocking, theTNF-alpha-supernatant mixtures were added and incubated for 2 h. ELISAswere developed using the biotinylated polyclonal rabbit anti-humanTNF-alpha antibody, for example, BAF210 (R&D Systems) at 0.2 ug/mlfollowed by mAvidin—HRP and then TMB.

None of the clones on M60 or M61 had any significant TNF-alphaneutralising activity (all clones demonstrated around or less than 10%TNF-alpha neutralisation). In contrast, adalimumab gave near completeinhibition in the ELISA on all plates where it was included. Many of theclones on M62 (derived from library 33) showed good TNF-alphaneutralisation (11 clones achieving 90% or greater neutralisation and 45clones achieving 50% or greater neutralisation). A few good neutralisingclones were also found on M63 (derived from library 35), but the numberswere much lower than from M62, even though the selection methods wereidentical (2 clones achieving 90% or greater neutralisation and 7 clonesachieving 50% or greater neutralisation).

2.2.3 Screening of Periplasmic Supernatants (PS) by Fixed ConcentrationL929 Cytotoxicity Assay (Master Plates M60-M63)

Materials

-   -   L929 cells (10000 cells/well)    -   human TNF-alpha fixed concentration for the assay: 500 pg/ml    -   Actinomycin D concentration: 0.75 ug/mL    -   PS from masterplates M60-M63 diluted in the assay 1:10    -   Pure anti-TNF-alpha VHH as positive control: 250 nM final        concentration    -   human TNF-alpha short dose-response curve: 500, 125, 31.25, 7.8        pg/ml    -   incubation times: 24h    -   resazurin cell viability reagent

Method

10000 cells/well in 100 ul were plated on day 0 in 96-well micro-platesin DMEM complete medium and incubated over night at 37 degrees C., 5%CO₂. On Day 1 dilution plates were set up (with volumes sufficient fortriplicates for each point) and in addition each plate contained ascontrols:

1. DMEM complete+0.01% Triton X-100

2. DMEM complete+0.75 ug/mL Actinomycin D

3. TNF-alpha dose-response curve+0.75 ug/mL Actinomycin D

The medium was removed from each well of the microplate and the cellswere incubated with 100 ul of DMEM complete containing hTNF-alpha at 500pg/mL+0.75 ug/mL Actinomycin D+PS (1:10) or with 100 ul of the differentcontrols. After 24 h of incubation at 37 degrees C. 5% CO₂, 10 ul ofresazurin was added to each well and the cells were incubated for 2 h at37 degrees C. 5% CO₂. 50 ul of 3% SDS was subsequently added to eachwell. The plates were then read on a fluorescent plate reader atExcitation (Ex) 544 nm/Emission (Em) 590 nm.

Results

None of the periplasmic supernatants of M60-61 showed a protectiveeffect on L929 cells against human TNF-alpha. The positive controlshowed protection against the effect of TNF-alpha, while the irrelevantVHH did not. Multiple periplasmic supernatants from the selected clonesof M62-63 showed a protective effect on L929 cells against TNF-alpha(107 clones achieved greater than or equal to 50% survival). Thepositive control showed TNF-alpha neutralisation (greater than or equalto 100%), while the irrelevant VHH showed less than 10% TNF-alphaneutralisation.

2.3 Evaluating TNF-Alpha Binding and Neutralising Activity (Master PlateM65)

ELISA and L929 cytotoxicity assays were performed on M65 in the samemanner as those described above in respect of M60-M63, unless statedotherwise.

2.3.1 Screening of Periplasmic Supernatants by ELISA (Master Plate M65)

M65 periplasmic supernatants of TNF-alpha specific variable domainclones (96 in total) were analysed for inhibition of TNFR-2-TNF-alphabinding by ELISA both immediately on thawing and after 18h incubation at37 degrees C.

ELISA plates were coated with 2 ug/ml, 50 ul/well etanercept at 4degrees C. overnight, washed and then blocked in preparation forincubation with the TNF-alpha-variable domain mixtures. adalimumab wasused at 10 nM as a positive control. The anti-TNF-alpha variable domainperiplasmic supernatants were initially diluted 1:10 in 1% BSA, thenmixed 1:1 with 2.5 ng/ml TNF-alpha and added to ELISA plates. Subsequentprocessing of ELISAs followed the protocol above described in respect ofM60-63.

Twenty eight of the supernatants gave greater than 80% inhibition ofTNF-alpha binding, of which 18 retained most or all inhibitory activityafter incubation at 37 degrees C.

2.3.2 Screening of Periplasmic Supernatants by L929 Cytotoxicity Assay(Master Plate M65)

The L929 cytotoxicity assay was performed with a total of 96 periplasmicsupernatants (PS) obtained from clones selected from 2 phage displaylibraries (M65—from #33NEW and #35NEW). The clones were picked afterperforming (i) a first round of selections on hsTNF-alpha (10 ug/mL)etanercept, and TEA for the elution and from (ii) a second round ofselections on cynomolgus sTNF-alpha (10 ug/mL) followed by completeelution with triethylamine. All the 96 PS were analysed by L929cytotoxicity assay using resazurin.

Materials

-   -   L929 cells (10000 cells/well)    -   human TNF-alpha fixed concentration for the assay: 500 pg/ml        final concentration    -   Dilution of PS in the assay: 1:10    -   Pure anti-TNF-alpha VHH as positive control: 250 nM final        concentration    -   M65 supernatants    -   TNF-alpha short dose-response curve: 500, 125, 7.8 pg/ml    -   incubation time: 22 h    -   Resazurin cell viability reagent

Method

10000 cells/well in 100 ul were plated on day 0 in 96 wells Costarmicro-plates in DMEM complete medium and incubated over night at 37degrees C. and 5% CO₂. On Day 1 dilution plates were set up (withvolumes sufficient for triplicates for each point) and in addition eachplate contained as controls:

1. DMEM complete+0.01% Triton X-100

2. DMEM complete+0.75 ug/mL Actinomycin D

3. TNF-alpha dose-response curve+0.75 ug/mL Actinomycin D

The medium was removed from each well of the microplate and the cellswere incubated with 100 ul of DMEM complete containing hTNF-alpha at 500pg/mL+0.75 ug/mL Actinomycin D+PS (1:10) or with 100 ul of the differentcontrols. After 22h of incubation at 37 degrees C., 10 ul of resazurinwas added to each well and the cells were incubated for 2 h at 37degrees C., 5% CO₂. 50 ul of 3% SDS were subsequently added to eachwell. The plates were then read using a fluorescent plate reader Ex544nm/Em590 nm.

Multiple periplasmic supernatants from the selected clones of M65 showeda protective effect on L929 cells against h-TNF-alpha. In 9 clones, thetreatment with the periplasmic fractions showed greater than 120%survival of L929 cells. A total of 53 clones out of 96 showed greaterthan 50% TNF-alpha neutralizing activity in the L929 assay and amongthem 32 showed nearly complete neutralization.

2.3.3 Resistance to Inactivation by Trypsin and Chymotrypsin

Supernatants (and purified samples) were tested for resistance of thevariable domain clones to inactivation by trypsin and chymotrypsin basedon the retention of their TNF-binding activity in the TNF-TNFR2 ELISA.

Method

Trypsin and chymotrypsin were dissolved separately (in half of the finalvolume) in 1 mM Tris-HCl pH 8.0, 20 mM CaCl₂) and then mixed together toobtain a final concentration of 3 mg/mL each (3 times the assayconcentration). 20 uL of each periplasmic extract was incubated with 10uL of PBS (not digested) or with 10 uL of 3 mg/mL trypsin+chymotrypsinmix (digested) for 75 min at 37 degrees C. 30 uL of ice-cold 2× proteasestop solution (2% BSA, 1×PBS, 5 mM NaETDA, 4 mM AEBSF, 0.6 μM Aprotinin,260 μM Bestatin, 2 mM EDTA, 28 μM E-64, 2 μM Leupeptin, 1 mM PBSF) wereadded to each sample, and subsequently 140 uL of 1×PBS, 1% BSA wereadded to each sample to reach a final 1:10 dilution of the periplasmicextracts. A range of concentrations including a top concentration of 3uM in 1% BSA was prepared for each purified sample. 10 uL of purifiedsample was incubated with 10 uL of trypsin and chymotrypsin mix (or PBSfor undigested sample) and 10 uL of trypsin/chymotrypsin buffer. Theperiplasmic and purified samples were stored over-night at −80 degreesC. and tested the next day in the TNFR-2-TNF-alpha ELISA assay. ELISAplates were coated with 0.7 ug/ml, 50 ul/well etanercept overnight,washed and then blocked in preparation for incubation with theTNF-alpha-variable domain samples. TNF-alpha variable domain sampleswere mixed 1:1 with 5 ng/ml h-TNF-alpha (to obtain a TNF-alpha assayconcentration of 2.5 ng/mL and 10 samples diluted 1:20) and 50 uL ofeach sample were added to the ELISA plates. After incubation at 4degrees C., ELISA plates were washed and incubated with 0.2 ug/mL ofbiotinylated polyclonal rabbit anti-h-TNF-alpha antibody for 1 h at RT.The ELISA plates were then washed, incubated with 1:1000modified-avidin-HRP conjugate (mAvidin-HRP) (mAvidin, for example,ExtrAvidin® (Sigma) is a modified form of egg white avidin that retainsthe high affinity and specificity of avidin for biotin, but does notexhibit the nonspecific binding at physiological pH that has beenreported for avidin) for 1 h at RT, washed again, and 100 uL of TMBELISA substrate were added to each sample. After 30 min the reaction wasstopped with 50 uL of 0.5M H2504 and the ELISA plates were read forabsorbance at 450 nm.

Results

11 variable domains (Q65B1-Q65F6) were from periplasmic supernatants and4 variable domains (Q62F2-Q62F11) were from purified samples. 10variable domains in periplasmic supernatant and 2 purified variabledomains were found to have good or excellent trypsin and chymotrypsinresistance. 10 of these variable domains were later found to belong tothe same family (Family 1, see part 2.4 below) and 2 of these variabledomains were later found to belong to the same family (Family 2, seepart 2.4 below). Q62F11, which belongs to neither Family 1 nor Family 2,was not protease resistant. The result with Q62F10 is thought to be dueto experimental error (FIG. 1).

2.4 Primary Evaluation Summary

M62 and M63: Of the 186 clones originally picked, 52 of the periplasmicsupernatants showed greater than or equal to 50% neutralisation of humansoluble TNF-alpha in the L929 cell cytotoxicity assay as well as greaterthan or equal to 50% inhibition of TNF-alpha-TNFR2 binding activity inthe ELISA assay. A sequence analysis performed on 25 of the variabledomain clones with greatest neutralising activities showed that thesecould be grouped into families. Representative clones were selected fromthe different family groups. The variable domain DNA sequences werere-cloned for high-level expression in E. coli and the purified variabledomains generated for more detailed evaluation studies.

M65: Of the 96 clones originally picked, 53 of the periplasmicsupernatants showed greater than 50% neutralising activity against humansoluble TNF-alpha in the L929 cell cytotoxicity assay as well as greaterthan or equal to 50% inhibition of TNF-alpha-TNFR2 binding activity inthe ELISA assay, with 32 showing nearly complete neutralization in bothassays. To identify the most stable clones, the periplasmic supernatants(96 in total) were analysed for inhibition of TNFR-2-TNF-alpha bindingby ELISA both immediately on thawing and after 18h incubation at 37degrees C. Eighteen of the supernatants retained most or all of theTNFR-2-TNF-alpha binding inhibitory activity. The most activeperiplasmic supernatants (28/96) were then selected to test forinhibition of cynomolgus monkey TNF-induced L929 cell cytotoxicity. Asequence analysis performed on 36 of the variable domain clones withgreatest TNF-alpha neutralising activities and resistance to proteasesshowed that these could be grouped with the M62 and M63 derived variabledomains into families. Representative M65 derived clones were selectedfrom the different family groups. The variable domain DNA sequences werere-cloned for expression in E. coli and the purified variable domainsgenerated for more detailed evaluation studies.

Example 3: Evaluation of Purified E. coli Recombinant Variable Domains

3.1 Production of Selected Variable Domain Clones in E. coli

DNA sequences of the variable domains selected from M62, M63 and M65were re-cloned for production in E. coli and then the expressed variabledomains were purified as follows.

Selected variable domains were subcloned from the phagemid vector intothe expression plasmid pMEK222 (pMEK222 is a gene3 deleted version ofthe phagemid pUR8100, and where the cloned variable domain is followedby c-myc and 6His tags, two stop codons and the M13 terminator sequence(see WO2013/064701)). The variable domain genes were digested with Sfiland Eco91l and ligated into pMEK222 cut with the same restrictionenzymes. E. coli strain BL21 DE3 was transformed by the ligations andplated on LB-agar plates supplemented with ampicillin and 2% glucose.Transformants were screened using colony PCR. Amplifications using theprimers M13.rev (SEQ ID NO: 81) and M13.fw (SEQ ID NO: 82) led to thegeneration of plasmids containing inserts of 700 bp and of ˜350 bp(empty plasmids) observed by PCR.

Variable domains were produced from pMEK222 by inoculation of a freshovernight grown culture at 1/100 dilution in 800 ml 2×YT, 0.1% glucoseand 100 ug/ml ampicillin and grown for 2h at 37 degrees C. Subsequently,1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) was added and theculture was grown for an additional 5 h at 37 degrees C. Bacteria wereharvested by centrifugation and resuspended into 30 mL PBS. Bacteriawere frozen by incubation at −20 degrees C. overnight. Bacteria werethawed at room temperature and fractionated by centrifugation. To thesoluble fraction, which contains the variable domain, Co²⁺ agarosebeads, for example, Talon resin (Thermo Scientific) were added to bindHis-tagged variable domain. After washing the beads, bound variabledomains were eluted with PBS supplemented with 150 mM imidazole.Finally, fractions containing the variable domains were dialyzed againstPBS to remove the imidazole.

TNF-alpha-neutralising activities of the purified variable domains wereevaluated in several in vitro assay systems to assess efficacy againstsoluble and membrane forms of human TNF-alpha and soluble cynomolgusmonkey TNF-alpha.

3.2 Inhibition of TNF-Alpha-Induced Cytotoxicity of L929 Cells

The assay of variable domains with TNF-alpha-neutralising activity wascarried out using an L929 murine cell line and a biological read-outbased on the induction of cytotoxicity by soluble human (or cynomolgusmonkey) TNF-alpha. L929 cells (10000 cells/well) were cultured for 24hin the presence of soluble TNF-alpha (500 pg/ml) and actinomycin (0.75ug/mL) together with dilutions of the purified variable domains. At theend of the experiment cytotoxicity was determined using resazurin. Theinhibition of soluble human TNF-induced cytotoxicity of mouse L929 cellswas tested to determine TNF-alpha neutralising activity of each of theE. coli recombinant 062, 063 and 065 series variable domains againsthuman and cynomolgus monkey TNF.

3.2.1 Purified Immunoglobulin Chain Variable Domains Selected from M62and M63

Materials

-   -   L929 cells (10000 cells/well)    -   Sterile polypropylene 96-well plates    -   h-TNF-alpha fixed concentration for the assay: 500 pg/ml    -   Actinomycin D concentration: 0.75 ug/mL    -   Purified variable domains (from MP 62-63) including Q62F2,        Q62F11, Q62E10, Q62F10    -   Range of dilutions of the purified variable domains: 300 nM-5 μM        (1:3 dilutions)    -   human-TNF-alpha dose-response curve: 3 ng/mL-0.5 pg/mL    -   Cynomolgus monkey TNF-alpha dose-response curve: 10 ng/mL-0.2        pg/mL    -   Adalimumab dose-response curve: 10 nM-0.5 μM    -   incubation times: 22 h    -   Resazurin cell viability reagent

Method

10000 cells/well in 100 ul were plated on day 0 in 96 wells micro-platesin DMEM complete medium and stored over night at 37 degrees C. and 5%CO₂. On Day 1 dilution plates were set up (with volumes sufficient fortriplicates for each point) and in addition the plates contained ascontrols:

1. DMEM complete+0.75 ug/mL Actinomycin D

2. DMEM complete+0.75 ug/mL Actinomycin D+0.5 ng/mL h-TNF-alpha

3. DMEM complete+0.01% Triton (only in the plate containingCyno-TNF-alpha dose 40 response)

4. DMEM complete (only in the plate containing Cyno-TNF-alpha,h-TNF-alpha and adalimumab dose responses)

The medium was removed from each well of the microplates and the cellswere incubated with 100 ul of each variable domain dilution or with 100ul of the different controls. After 22 h of incubation at 37 degrees C.and 5% CO₂, 10 ul of resazurin were added to each well and the cellswere incubated for 2 h at 37 degrees C. 50 ul of 3% SDS weresubsequently added to each well. The plates were then read using afluorescent plate reader Ex544 nm/Em590 nm.

3.2.2 Purified Immunoglobulin Chain Variable Domains Selected from M65

Materials

-   -   L929 cells (10000 cells/well)    -   Sterile polypropylene 96-well plates    -   DMEM    -   Human TNF-alpha concentration: 500 pg/ml    -   Cynomolgus monkey TNF-alpha concentration: 500 pg/ml    -   Actinomycin D concentration: 0.75 ug/mL    -   Purified variable domains from M65 including Q65D1, Q65D3,        Q65B1, Q65C7, Q65A3, Q65E12, Q65F6    -   Range of dilutions of the purified variable domains: 300 nM-5 μM        (1:3 dilutions)    -   Human TNF-alpha and Cynomolgus TNF-alpha dose-response curves:        10 ng/M1-0.5 pg/mL    -   Adalimumab dose-response curve: 10 nM-0.5 μM    -   incubation times: 22h    -   Resazurin cell viability reagent

Method

10000 cells/well in 100 ul were plated on day 0 in 96 wells micro-platesin DMEM complete medium and stored over night at 37 degrees C. and 5%CO₂. On day 1 serial dilutions 1:3 (in DMEM+Act.D+TNF) for each purifiedvariable domain were set up (with volumes sufficient for triplicates foreach point) starting from a top concentration of 300 nM. For adalimumabthe top concentration used was 10 nM.

The following controls were added to the plates:

1. DMEM complete+0.75 ug/mL Actinomycin D

2. DMEM complete+0.75 ug/mL Actinomycin D+0.5 ng/mL of h-TNF-alpha orcyno-TNF-alpha

3. DMEM complete+0.01% Triton (only in the plate containing theTNF-alpha dose responses)

4. DMEM complete (only in the plates containing the TNF-alpha doseresponses)

The medium was removed from each well of the microplates and the cellswere incubated with 100 ul of each variable domain dilution or with 100ul of the different controls. After 22 h of incubation at 37 degrees C.and 5% CO₂, 10 ul of resazurin was added to each well and the cells wereincubated for 2 h at 37 degrees C. 50 ul of 3% SDS was subsequentlyadded to each well. The plates were then read using a fluorescent platereader Ex544 nm/Em590 nm.

3.2.3 Results of L929 Cytotoxicity Assays

TABLE 1(a) Anti-TNF-alpha clones derived from M62 and M63: inhibition ofhuman and cynomolgus TNF-induced L929 cell cytotoxicity Inhibition ofhuman and cynomolgus TNF- induced L929 cell cytotoxicity EC50 (nM) CloneID Family Human TNF Cynomolgus TNF Q62F2 1 1 0.8 Q62F11 — 6 1.5 Q62E10 20.2 <0.1 Q62F10 2 2 1.3

TABLE 1(b) Anti-TNF-alpha clones derived from M65: inhibition of humanand cynomolgus TNF-induced L929 cell cytotoxicity Inhibition of humanand cynomolgus TNF- induced L929 cell cytotoxicity EC50 (nM) Clone IDFamily Human TNF Cynomolgus TNF Q65D3 1 0.15 0.1 Q65D1 1 0.1 0.06 Q65B11 0.04 0.01 Q65C7 1 0.15 0.03 Q65A3 2 0.15 0.04 Q65E12 2 4 4 Q65F6 2 1.51 Adalimumab 0.1 0.04

It can be seen that variable domains belonging to Family 1 generallyshowed extremely effective inhibition of both human and cynomolgusTNF-alpha-induced cytotoxicity and variable domains belonging to Family2 also generally showed very effective inhibition of both human andcynomolgus TNF-alpha-induced cytotoxicity. A number of variable domainsshowed similar performance to adalimumab.

3.3 Inhibition of TNF-TNFR2 Binding Activity (Purified ImmunoglobulinChain Variable Domains Selected from M65)

Maxisorb plates were coated with etanercept (50 ul/well of 0.7 ug/ml).Variable domains were diluted and mixed with either human or cynomolgusmonkey TNF-alpha (1.25 ng/ml) to allow binding before adding to theELISA plates. TNF-alpha bound to etanercept was detected withbiotinylated rabbit polyclonal anti-TNF-alpha antibody (0.2 ug/ml) andmAvidin-HRP followed by TMB and the level of competition by the variabledomain for TN F-alpha binding to etanercept was determined. Particularvariable domains were capable of binding to human and cynomolgus monkeyTNF-alpha leading to inhibition of TNF-alpha-TNFR2 interactions.

3.3.1 Results of TNF-TNFR2 Binding Activity Assays

TABLE 2a Anti-TNF-alpha clones derived from M62 and M63 - inhibition ofhuman and cynomolgus TNF-alpha binding to TNFR2 Inhibition ofTNF-alpha-TNFR2 binding (ELISA) EC50 (nM) Clone ID Family Human TNFCynomolgus TNF Q62F2 1 0.7 1.3 Q62F11 — 2.3 1.4 Q62E10 2 0.8 1.2 Q62F102 30 108 Adalimumab — 0.3

TABLE 2b Anti-TNF-alpha clones derived from M65 - inhibition of humanand cynomolgus TNF-alpha binding to TNFR2 Inhibition of TNF-TNFR2Binding (ELISA) EC50 (nM) Clone ID Family Human TNF Cynomolgus TNF Q65D31 0.33 0.45 Q65D1 1 0.37 0.32 Q65B1 1 0.29 0.45 Q6507 1 0.35 0.32 Q65A32 1.3 1.1 Q65E12 2 47 65 Q65F6 2 13 24

It can be seen that variable domains belonging to Family 1 generallyshowed extremely effective inhibition of both human and cynomolgusTNF-alpha-TNFR2 binding and variable domains belonging to Family 2 alsogenerally showed very effective inhibition of both human and cynomolgusTNF-alpha-TNFR2 binding. A number of variable domains showed similar orbetter performance to adalimumab.

3.4 Inhibition of Soluble TNF-Alpha-Induced Activation of NF-kB/SEAPHEK-293 Reporter Cells

The potencies of selected variable domains were measured initially basedon the inhibition of cytotoxicity of soluble TNF-alpha towards murineL929 cells. To confirm that these variable domains were also effectiveand potent in an assay using human cells, experiments were performedusing HEK-293-NF-kB-SEAP reporter cells. NF-kB/SEAP HEK-293 cells arestably transfected with the SEAP (secreted alkaline phosphatase)reporter gene under the transcriptional control of an NF-kB responseelement. Induction of the reporter gene by soluble human TNF-alpha wasconfirmed and the ability to inhibit this response byTNF-alpha-neutralising antibodies was demonstrated. In these cellsactivation of the SEAP reporter gene is dependent on the NF-kB pathway,which is activated by soluble TNF-alpha via cell membrane TNFR1receptors.

In the first experiment Q65B1 (Family 1), Q65C7 (Family 1), Q62E10(Family 2) and adalimumab were tested. In the second experiment Q65D3was compared to Q65B1.

3.4.1 Inhibition by Q65B1, Q65C7, Q62E10 and Adalimumab

Materials

-   -   HEK-293 NF-kB/SEAP cells (Imgenex): 3.5×10⁴ cells/well, 2×104        cells/well (ii), 10⁴ cells/well (ii)    -   sterile 96-well plates    -   DMEM (Sigma, D6429) supplemented with 10% FBS, Pen/Strep, and        500 ug/mL Geneticin antibiotic (G418, Invitrogen, 10131027)    -   Human TNF-alpha (Invitrogen, PHC3015) concentration: 2 ng/ml    -   Purified immunoglobulin chain variable domains Q65B1, Q65C7,        Q65D3, Q65E5, Q65E6 and Q62E10    -   Range of dilutions of immunoglobulin chain variable domains and        adalimumab: 500 Nm-2 μM; 150 nM-1 μM; (3.5 fold dilutions)    -   Human TNF-alpha dose-response curves: 10 ng/mL-0.01 ng/mL    -   Controls in the plates:    -   cell medium (CM)    -   human soluble TNF-alpha (“hs-TNF-alpha”) at 2 ng/ml    -   0.01% Triton X-100 (for TNF dose response)    -   Incubation times: 22.5h    -   QuantiBlue medium (InvivoGen): 200 uL    -   Supernatant volume tested: (i) 10 uL, (ii) 10 uL and 20 uL    -   Time-point reading: 2h 45 min    -   BioRad Plate reader (620 nm)

Methods

3.5×10⁴ cells/well of NF-kB/SEAP HEK-293 cells were plated in 100 uL onday 0 in 96-well sterile flat bottom micro-plates and stored over nightat 37 degrees C. and 5% CO₂. On day 1 serial dilutions (3.5 fold) foreach purified immunoglobulin chain variable domain were set up (withvolumes sufficient for triplicates) in 100 uL of HEK medium containing 2ng/mL hs-TNF-alpha. The medium was removed from the assay plates andsubstituted with 100 uL of each sample dilutions. After 22.5h at 37degrees C. and 5% CO₂, 10 uL or 20 uL of each supernatant were mixedwith 200 uL of pre-warmed Quanti Blue medium (InvivoGen) in a 96 wellflat bottom NUNC plate. Following 2h 45 min of incubation at 37 degreesC. in dark with shaking, the SEAP production was measured with theBioRad plate reader at 620 nm.

Results showed that Q65B1 (Family 1), Q65C7 (Family 1) and Q62E10(Family 2), achieved maximal inhibition of induced cell activationinduced by soluble human TNF-alpha. Results showed that theseimmunoglobulin chain variable domains potently and effectively inhibitresponses to soluble TNF-alpha mediated via TNFRs expressed on humancells (FIG. 2A).

3.4.2 Inhibition by Q65D3 and Q65B1 (Both Family 1)

Materials and methods were as described above under point 3.4.1, exceptfor different numbers of cells/well (3.5×10⁴, 2×10⁴, and 1×10⁴) andvolumes of cell supernatant after stimulation with TNF (10 uL and 20uL).

Results

Both Q65B1 and Q65D3 are able to completely neutralize the activity ofhuman soluble TNF-alpha on the HEK-293 NF-kB/SEAP reporter cells, withEC50 values of around 0.2 nM for Q65B1 and 0.6 nM for Q65D3 (FIG. 2B).

3.5 Inhibition of Membrane TNF-Alpha Induced Cell Activation

Both soluble and membrane-bound TNF-alpha can induce activation of cellsexpressing TNF-alpha-receptors and both forms are considered tocontribute to inflammation and pathology in Crohn's disease. Variabledomains capable of binding and neutralising both soluble andmembrane-bound TNF-alpha activity are likely to be the most effective.

A cell contact reporter assay was developed to ensure that variabledomains could be identified with the ability to inhibitmembrane-TNF-alpha induced cell activation. A CHO cell line was createdthat constitutively expresses a non-cleavable transmembrane form ofhuman TNF-alpha (mTNF-alpha-CHO). In co-culture with the NF-kB/SEAPHEK-293 cells, the mTNF-alpha-CHO cells induced activation of thereporter gene. The ability of the different variable domains to inhibitmTNF-alpha induced cell activation could therefore be determined.

3.5.1 Results of Membrane TNF-Alpha Induced Cell Activation Assays

TABLE 3a Anti-TNF-alpha clones derived from M62 and M63 - inhibition ofmTNF-alpha-induced NF-kB/SEAP HEK-293 reporter cell activationInhibition of mTNF-alpha-induced Clone ID Family HEK-NF-kB SEAP Reporter(EC50 = nM) Q62F2 1 34 Q62F11 — 40 Q62E10 2 80 Q62F10 2 — Adalimumab 1-3

TABLE 3b Clones derived from M65 - inhibition of mTNF-alpha-inducedHEK-NF-kB SEAP reporter cell activation Inhibition of mTNF-alpha-inducedClone ID Family HEK-NF-kB SEAP Reporter (EC50 = nM) Q65D3 1 16.0 Q65D1 115.3 Q65B1 1 10.9 Q65C7 1 14.1 Q65A3 2 23.9 Q65E12 2 173.8 Q65F6 2 109.6Adalimumab 1.2

It can be seen that variable domains belonging to Family 1 generallyshowed effective inhibition of mTNF-alpha and variable domains belongingto Family 2 also generally showed inhibition of mTNF-alpha.

Example 4: Cross-Reactivity with Adalimumab

Adalimumab 1 μg/ml was coated onto HPM plates then incubated with humanTNF-alpha in the absence or presence of the different variable domains.After washing to remove free TNF-alpha-variable domain complexes,TNF-alpha remaining bound to the immobilised mAb was detected byincubation with biotinylated rabbit polyclonal anti hTNF-alpha antibody;mAvidin-HRP followed by TMB were then added for ELISA colourdevelopment.

Results

Q62E10 (Family 2), Q62F2 (Family 1), Q62F10 (Family 2), Q65A3 (Family2), Q65B1 (Family 1), Q65C7 (Family 1), Q65D1 (Family 1), Q65D3 (Family1), Q65E12 (Family 2), Q65F6 (Family 2) and Q62F11 (neither Family 1 norFamily 2), showed complete inhibition of TNF-alpha binding toadalimumab.

Example 5: Stability of Purified E. coli Recombinant Anti-TNF-AlphaImmunoglobulin Chain Variable Domains in Supernatant from Mouse SmallIntestine

The purified variable domains were tested in the presence of asupernatant extract prepared from the pooled small intestinal contentsof several mice. Following incubation of the variable domains in thepresence of the intestinal extract for different time periods,TNF-alpha-binding activities of the “digested” variable domain sampleswere assayed using the h-TNF-alpha-TNFR2 ELISA.

Mouse Small Intestine Supernatants

Mouse small intestines were excised, the solid contents removed and theintestines were rinsed with 1 ml of 0.9% saline. The solid and liquidparts were combined in 1.5 mL microfuge tubes, vortexed to homogeny andcentrifuged at 16k×rcf for 10 minutes at 4 degrees C. The supernatantswere removed and stored at −80 degrees C. before use.

Plates and Antigens

The anti-TN F-alpha variable domains were assayed on HPM plates coatedwith 1 ug/mL etanercept (50 uL/well) overnight at 4 degrees. The plateswere blocked with 1% BSA for a minimum of 1 hour before use in theassay.

Protease Stop Solution

Protease inhibitor cocktail tabs were dissolved in 5 mL protease stopbuffer (2% BSA, 1×PBS, 5 mM NaEDTA) to make a 20× protease inhibitorstock solution. A 2× protease stop solution (as described in section2.3.3) was produced by diluting the 20× protease inhibitor solution 1 in10 with a 1 in 100 dilution of PMSF in protease stop buffer

Methods

Digests

Variable domain stock solutions were prepared in 0.4% BSA at 250 ug/mL.To 55.2 uL of SI supernatant on ice, 4.8 ul of variable domain was addedand mixed by vortexing. 25 uL was immediately removed and mixed with 25uL of ice-cold protease stop solution (undigested control), and frozenat −80 degrees C. A second aliquot of 25 ul was moved to a single wellin a polycarbonate thin-walled PCR plate and incubated for 1 to 24 hoursat 37 degrees C. (digested variable domain). After incubation, thedigested variable domain samples were placed on ice and 25 uL ofice-cold protease stop solution was added to each tube. The samples werefrozen at −80 degrees C. before assay.

ELISA

The variable domains were diluted in 1% BSA+1× protease inhibitorsolution, made by diluting 20× protease inhibitor stock solution in 1%BSA, and mixed 1:1 with 5 ng/mL h-TNF-alpha. TNF-alpha mix wastransferred to the blocked HPM ELISA plates coated with 1 ug/mLetanercept. The plates were incubated for 2 h washed 4× with PBS 0.5%PEG20 (PBST), dried and then incubated with biotinylated rabbitpolyclonal anti-human TN F-alpha antibody (50 uL/well, 0.27 ug/mL) for 1hour with shaking at RT. After 1h, the plates were washed 4× with PBST,dried by tapping and then incubated with mAvidin-HRP (50 uL/well, 1/1000dilution) with shaking at RT for 30 min. The plates were then washedwith PBST 4×, dried by tapping and developed using 100 uL TMB. Standardcurves of the variable domains (in PBS) were run alongside thedigested/non-digested samples. The top concentration of the variabledomains used in the standard curve and test samples was 300 ng/mL and200 ng/mL respectively.

Data Analyses

The level of inhibition achieved by the variable domain was calculatedby subtracting the signal in the digested and undigested-controlvariable domains from a TNF-only control (maximal signal). This gave thesignal reduction for each variable domain, which is proportional to theamount of variable domain in the sample. The % survival was calculatedby dividing the signal reduction of the digested variable domain by thesignal reduction of the non-digested variable domain for a singledilution.

Results

62F2 and 65B1 (both Family 1) showed greater than 80% survival, 62E10showed greater than 70% survival (Family 2), 65D1 and 65C7 (Family 1)showed around 50% survival.

A time course study was then performed to measure performance at 1h, 3hand 7 h. The results broadly agreed with the previous small intestinalsupernatant work.

Example 6: Optimisation of Selected Anti-TNF-Alpha Immunoglobulin ChainVariable Domains

Variable domains investigated so far have not been mutated and purifiedvariable domains have all included C-terminal tags (c-myc-6His orFlag-6His). Mutants were designed to explore whether the modification ofN-terminal amino acids, or absence of C-terminal-tags, when combinedwith mutations to Q65B1, would lead to further increased stability.

6.1 Constructs and Mutations

(i) A set of Q65B1 monomer mutants (ID7F-EV, ID8F-EV ID9F-EV, ID13F-EV,ID14F-EV and ID15F-EV; all including His-tags) were designed toinvestigate the effects of particular mutations on protease resistance.

(ii) A set of bihead constructs (ID22F-ID29F) were designed whichincluded mutated Q65B1 monomer variants and removing His-tags.

(iii) A set of Q65B1 monomer mutants were designed which includedmutating the beginning of the sequence to DVQLV and removing His-tags toinvestigate possibilities for future yeast expression optimisation(DVQLV mutants ID37F and ID38F compared to EVQLV equivalents ID32F(ID32F is ID8F-EV without His-tag) and ID34F, respectively).

Further details of these constructs and mutations are shown in thetables below. Mutations are shown underlined and emboldened relative tothe parent Q65B1 monomer sequence.

Details of Constructs and Mutations

FR1 CDR1 FR2 CDR2 ID7F-EV EVQLVESGGGLVQPG A SLKLSCAASGFDFS  SHWMYWVRQAPGKELEWLS EINTNGLITKYGDSVKG ID8F-EV EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKG ID9F-EVEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSV H GID13F-EV EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLSEINTNGLITKYGDSVKG ID14F-EV EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMYWVRQAPGKELEWLS EINTNGLITKYGDSVKG ID15F-EV EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKG ID22FEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMYWVRQAPGKELEWLS EINTNGLITKYGDSVKG ID23F EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKG GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKGID24F EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT HYGDSVKG GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKG ID25FEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMYWVRQAPGKELEWLS EINTNGLIT H YGDSVKG ID26F EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKG GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSVKGID27F EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT HYGDSV H G GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT H YGDSV H G ID28FEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSV H GGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFDFS SSHWMYWVRQAPGKELEWLS EINTNGLITKYGDSV H G ID29F EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSV H G GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSV H GID34F EVQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLS EINTNGLIT HYGDSVKG ID37F D VQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMY WVRQAPGKELEWLSEINTNGLIT H YGDSVKG ID38F D VQLVESGGGLVQPGGSLKLSCAASGFDFS  SHWMYWVRQAPGKELEWLS EINTNGLIT H YGDSVKG FR3 CDR3 FR4 ID7F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID8F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID9F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID13F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS ID14F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN H GQGTQVTVSS ID15F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN H GQGTQVTVSS ID22FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID23FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID24F RFTVSRNNAAN SMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS RFTVSRNNAAN SMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID25F RFTVSRNNAAN SMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS RFTVSRNNAAN SMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS ID26FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS ID27FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGT L VTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGT L VTVSS ID28FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID29FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSSRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS ID34FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS ID37FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID38FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ H GLN KGQGTQVTVSS

6.2.1 Effects on Potency and Resistance to Inte80-Stinal Proteases

(i) ID7F-EV, ID8F-EV ID9F-EV, ID13F-EV, ID14F-EV and ID15F-EV

Naïve mouse small intestinal supernatant: the contents of the smallintestines from seven C57BL/6 male mice were removed with 0.9% saline,combined, homogenised and centrifuged. The resulting supernatant wasremoved, aliquoted and frozen.

Human Faecal Supernatant Pool: the faecal samples were turned intoslurries with addition of 1×PBS. The slurries were then pooled,centrifuged and the supernatants removed, aliquoted and stored at −80degrees C. This process removes the faecal matrix, including anycellular material.

The anti-TNF-alpha variable domains were assayed on HPM plates coatedwith 1 ug/mL etanercept (50 unwell), overnight. The plates were blockedwith 1% BSA for a minimum of 1 hour before use in the assay. A 2×protease stop solution was produced as described in Example 5.

Digests

Variable domain stock solutions at 250 ug/mL were prepared in 0.34%(3400 ug/mL) BSA. To 55.2 uL of faecal or small intestinal supernatanton ice, 4.8 ul of variable domain was added and mixed by vortexing. 25uL was immediately removed and mixed with 25 uL of ice-cold proteasestop solution (undigested control), and frozen at −80 degrees C.Aliquots of 25 ul were placed in wells of a polycarbonate thin-walledPCR plate and incubated for 17 h or 7 h respectively at 37 degrees C.After incubation, the digested variable domain samples were placed onice and 25 uL of ice-cold protease stop solution was added to each tube.The samples were frozen at −80 degrees C. before assay.

ELISA

The variable domains were diluted in 1% BSA+1× protease inhibitionsolution as described in Example 5, mixed 1:1 with 5 ng/mL h-TNF-alpha,and incubated at RT for 1 h. The variable domain TN F-alpha mixture wasthen loaded onto blocked ELISA plates coated with 1 ug/mL etanercept andincubated for 2 hours with shaking at RT. The plates were washed 4× withPBST, dried by tapping and incubated with the biotinylated rabbit antihuman-TNF-alpha polyclonal antibody (50 uL/well, 0.3 ug/mL) for 1 hourwith shaking at RT. After 1h, the plates were washed and as before andincubated with mAvidin-HRP (50 uL/well, 1/1000 dilution) with shaking atRT for 30 min. The plates were then washed and dried as before anddeveloped using 100 uL TMB. Standard curves of the variable domains (inPBS) were run alongside the digested/non-digested samples. The topconcentration of the variable domains used in the standard curve andtest samples was 100 ng/mL.

Data Analyses

After digestion, the variable domain concentrations were measured usingthe TNFR2-interference ELISA. In this assay, the variable domains aremixed with TNF-alpha. The remaining level of TNF-alpha is then measured.The concentration of variable domain is inferred from the amount ofTNF-alpha inhibited from binding the TNFR2 receptor that is bound to theELISA plate. Raw OD450 values were adjusted with blank readings takenfrom wells containing 1% BSA only. Standard curves were plotted usingGraphpad Prism using nonlinear regression to fit four-parameter curves.variable domain concentrations in the test samples were calculated inGraphpad Prism using the standard curve. The % survival was calculatedby dividing the average variable domain concentration in the 0-timepoint wells by the average variable domain concentration for a singletime point. The standard error of the ratio of two means was calculated.

Results

Evaluation of the Q65B1 variable domain monomers showed that inclusionof these mutations surprisingly did not greatly affect TNF-alphaneutralisation potency (TNF-alpha-TNFR2 ELISA) of the modified variabledomains.

ID8F-EV and ID13F-EV showed improved resistance to inactivation byproteases present in mouse intestinal and human faecal supernatantextracts (FIG. 3).

(ii) Constructs ID22F-ID29F

Materials

-   -   HEK-293 NF-kB/SEAP cells: 10⁴ cells/well in 50 uL    -   96-well plates    -   DMEM supplemented with 10% FBS, Pen/Strep, and 500 ug/mL        Geneticin antibiotic (G418)    -   Human TN F-alpha concentration: 2 ng/ml    -   Purified homo bi-head variable domains: ID22F-ID29F    -   Variable domain dilutions: 20 nM-2.1 μM; 2.5 fold dilutions    -   Incubation time: 23h    -   SEAP colourmetric substrate medium: 200 uL; supernatant volume        tested: 20 uL; time-point reading: 2h    -   Plate reader (620 nm)

Methods

10⁴ cells/well of NF-kB/SEAP HEK-293 cells were plated in 50 uL in96-well flat bottom microplates and incubated overnight at 37 degrees C.and 5% CO₂. The following day serial dilutions (2.5 fold) for eachpurified variable domain were set up at double the assay concentration(with volumes sufficient for triplicates) in 50 uL of HEK mediumcontaining 4 ng/mL hs-TNF-alpha. Variable domains and TNF-alpha wereincubated for 1 h at RT and shaken before adding 50 uL of each dilutionto the assay plates. After 23 h at 37 degrees C. and 5% CO₂, 20 uL ofeach supernatant were mixed with 200 uL of pre-warmed SEAP medium in 96well flat bottom plates. Following 2 h incubation at 37 degrees C. inthe dark with shaking, the SEAP production was measured with a platereader at absorbance 620 nm.

Results

The Q65B1 homo-bihead derivatives (including those with changes K59H,K65H, K78S, K101H) were equally potent and effective with EC50 valuesbetween 0.02 nM-0.03 nM in the soluble TNF-induced HEK293-NF-kB-SEAPreporter assay. Potency of the Q65B1 homo bihead was increasedapproximately 10-fold relative to the monomer.

(iii) ID32F, ID34F, ID37F, ID38F

To avoid the possibility of generating a product with a cyclisedN-terminal glutamate if expressed in yeast, the effects of changing theN-terminal amino acid of the Q65B1-based variable domain from a glutamicacid to an aspartic acid residue (which is not susceptible tocyclisation) was investigated. Mutants were generated of Q65B1 toproduce the corresponding Q65B1 with N-terminal D residue and variantscombining each of these with the favourable protease resistancemutations.

sTNF-Alpha Neutralising Ability of ID-32F, ID-34F, ID-37F, and ID-38FUsing NF-kB/SEAP HEK-293 Cell Reporter Assay

To confirm that ID-38F and ID-34F neutralize soluble human TNF-alpha,these variable domains were tested in the NF-kB/SEAP HEK-293 cellularreporter assay in comparison to the commercial anti-TNF-alpha antibodyinfliximab (Remicade). Results were obtained from two separateexperiments performed on different days.

Materials

-   -   HEK-293 NF-kB/SEAP cells (Imgenex): 10⁴ cells/well in 50 uL    -   sterilised 96-well plates    -   DMEM (Sigma, D6429) supplemented with 10% FBS, Pen/Strep, and        500 ug/mL Geneticin antibiotic (G418, Invitrogen, 10131027)    -   Human TN F-alpha (Invitrogen, PHC3015) concentration: 2 ng/ml    -   5 purified variable domains: ID-32F, ID-34F, ID-37F, ID-38F and        Q65B1    -   infliximab dilution: 10 nM-1 μM (1st exp); 2 nM-1 μM (2nd exp)    -   Variable domain dilutions: 10 nM-1 μM (1st exp); 5 nM-1 μM (2nd        exp)    -   Incubation time: 22h    -   QuantiBlue medium (InvivoGen): 200 uL; supernatant volume        tested: 20 uL; time-point reading: 2h    -   BioRad Plate reader (620 nm)

Methods

10⁴ cells/well of NF-kB/SEAP HEK-293 cells were plated in 50 uL in96-well sterilised flat bottom micro-plates and incubated over night at37 degrees C. and 5% CO₂. The following day serial dilutions of variabledomains and infliximab (2.5 fold) were set-up at double the assayconcentration in HEK medium containing 4 ng/mL hs-TNF-alpha (for Exp 1).In the second experiment serial dilutions (1.9 fold for the variabledomain and 2.2 fold for adalimumab) were set up at 4× the assayconcentration in 120 uL of HEK medium. 100 uL of each dilution were thendiluted 1:1 with 100 uL of HEK medium containing 8 ng/mL hsTNF-alpha(4×) to have variable domains and TN F-alpha at double the assayconcentrations. Variable domains and TNF were incubated for 1h at RTshaking before adding 50 uL of each dilution to the assay plates. After22h incubation at 37 degrees C. and 5% CO₂, 20 uL of each supernatantwere mixed with 200 uL of pre-warmed Quanti Blue medium in 96 well flatbottom NUNC plates. Following 2h incubation at 37 degrees C. in darkwith shaking, the SEAP production was measured with the BioRad platereader at 620 nm.

Results

The results of experiment 1 are shown in FIG. 4A and the results ofexperiment 2 are shown in FIGS. 4B and 4C. All the variable domains showcomplete sTNF-alpha neutralisation and similar potencies with EC50values between 0.3 nM and 0.5 nM. It is clear that the addition of themutations in these variable domains does not affect the potency of thesevariable domains in neutralising sTNF-alpha. Infliximab does not give acomplete sTNF-alpha neutralization in either of the experiments showingnearly plateaued dose-response curves with ˜80% maximum neutralizationat roughly 2 nM.

mTNF-Alpha Neutralising Activity of ID-34F, ID-38F, Q65B1, ID-8FEV,Adalimumab, and Infliximab Using NF-kB/SEAP HEK-293 Cellular ReporterAssay

To confirm that ID-38F and ID-34F neutralize membrane bound TN F-alpha,these variable domains were tested in the NF-kB/SEAP HEK-293 cellularreporter assay in comparison to 2 progenitors (Q65B1 and ID8F-EV). 2commercial anti-TNF-alpha antibodies (adalimumab and infliximab) weretested in the same assay for reference.

Materials:

-   -   NF-kB/SEAP HEK-293 cells (Imgenex) concentration: 3.5×10⁴        cells/well    -   Stable TNF-alpha_DEL expressing Flp-ln CHO Cell Line (Invitrogen        & GeneArt, Life Technogies): 25×103 cells/well    -   2 purified anti-TNF-alpha mutant variable domains: ID-34F and        ID-38F    -   2 purified anti-TNF-alpha variable domains: Q65B1 and ID8F-EV    -   2 antibodies: adalimumab and infliximab    -   Variable domain/Ab dilutions: 300 nM-0.11 nM (2.2 fold        dilutions)    -   Incubation time: 24h    -   QuantiBlue medium (InvivoGen): 200 uL; supernatant volume        tested: 10 uL; time-point reading: 2h    -   BioRad Plate reader (620 nm)

Methods:

3.5×10⁴ cells/well of NF-kB/SEAP HEK-293 cells in 50 uL were plated onday 0 in 96-wells flat bottom micro-plates and stored over night at 37degrees C. and 5% CO₂. On day 1 serial dilutions for each purifiedvariable domain were set up (with volumes sufficient for triplicates) atthree folds the assay concentrations in 50 uL of HEK medium. 50 uL ofeach variable domain dilution (3×) were added to the assay plates andthe plates were then stored at 37 degrees C. and 5% CO₂ for 1h. 50 uL ofm-TNF-alpha CHO cells (25000 cells/50 uL) prepared in HEK medium wereadded to the HEK-assay plates to have the final variable domain assayconcentrations (1×). The cells were incubated for 24h at 37 degrees C.and 5% CO₂. On day 2, 10 uL of each supernatant were mixed with 200 uLof pre-warmed Quanti Blue medium (InvivoGen). After 2h incubation at 37degrees C. in dark with shaking, the SEAP production was measured usingthe BioRad plate reader at 620 nm. The resultant dose response curveswere analysed by GraphPad Prism software.

Results

TABLE 4 mTNF-alpha neutralisation by anti-TNF-alpha variable domains andcommercial anti-TNF-alpha antibodies Variable domain/ antibody EC50 (nM)ID-34F 4.71 ID-38F 5.16 Q65B1 8.72 ID8F-EV 7.69 Adalimumab 3.14Infliximab 3.79

ID-34F and ID-38F show similar potencies in neutralizing themembrane-bound TNF-alpha indicating that changing the N-terminal aminoacid of the variable domain from E to D does not affect the potency ofthe variable domain. ID-34F and ID-38F seem to be slightly more potentthan the parental monomers Q65B1 and ID-8FEV against mTNF. ID-34F andID-38F are only marginally less potent than the commercial antibodiesadalimumab and infliximab in neutralizing mTNF-alpha.

TNFR1 ELISA Comparison of ID38F

As TNFR1 is also thought to play a role in TNF-alpha pathology, an ELISAwas performed to test the abilities of ID38F and adalimumab inpreventing binding of TNF to TNFR1.

Method

A sterile 96-well microtitre plate was coated with 50 ul 0.5 ug/mlhTNFR1 (R&D Systems) per well overnight at 4 degrees C. Plate was washedin PBST using a plate washer and blocked for 1 hour in 200 ul 1% BSA perwell. A threefold dilution series beginning at 30 nM for each antibodywas incubated with 2.5 ng/ml TNF-alpha for 1 hour, 50 ul/well of whichwas then added to the washed plate and incubated for 5 hours. The platewas washed and incubated with 50 ul/well of a 1/1000 dilution ofbiotinylated rabbit alpha-hTNF (Peprotech P31AB7) in 1% BSA andincubated overnight at 4 degrees C. The plate was washed and incubatedwith 50 ul/well of a 1/1000 dilution of mAvidin HRP in 1% BSA for 1 hourbefore a final wash and incubation with 100 ul/well TMB substrate. Thereaction was stopped after 20 minutes with 50 ul/well 0.5 M H2504 andabsorbance was read at 450 nm. EC50 values were calculated from ELISAdata in GraphPad Prism.

Results

TABLE 5 Anti-TNF agent EC50 (pM) ID38F 610.0 Adalimumab 200.5

ID38F is able to neutralise binding of TN F-alpha to TNFR1 at asub-nanomolar EC50. Higher potency of adalimumab in this assay mayreflect the fact that this molecule is divalent while ID38F ismonovalent.

TNFR2 ELISA and Stability Assays

Evaluation of the TNF-alpha-binding activities of the variable domainmonomers in the TNFR2 ELISA showed that changing the N-terminal aminoacid of the variable domain from E to D had no significant effect.

Stabilities of the variable domain monomers were tested as previously bymeasuring their resistance to inactivation by proteases present in mousesmall intestinal and human faecal supernatants. Changing the N-terminalamino acid of the variable domain from E to D had only a very slighteffect on protease stability.

6.2.2 Summary

These results have shown that a monomer related to Q65B1, with theN-terminal sequence DVQLV and amino acid changes K59H and K101H(“ID38F”), in particular, has potent TNF-alpha-neutralising activity,excellent resistance to inactivation by small intestinal and faecalproteases and the potential for production of a product that is notsusceptible to pyroglutamation if it were to be expressed in yeast.

Example 7: Stability to Intestinal and IBD Inflammatory Proteases

7.1 Evidence of Stability to Proteases Present in Inflamed IBD Tissue

Protease activities that can lead to rapid degradation of therapeuticantibodies are up-regulated in Crohn's disease (MMP3, MMP12, cathepsin)(Biancheri et al ECCO, Dublin 2011 Abstract P007, herein incorporated byreference in its entirety)). It was therefore important to investigatethe stabilities of anti-TN F-alpha variable domains in the presence ofthe purified inflammatory proteases and in more complex IBD mucosaltissue lysates.

Recombinant human MMP-3 and recombinant human MMP-12 were obtained fromcommercial suppliers for example, R&D systems (513-MP-010 and 917-MP-010respectively). Enzyme activations and 22-hour incubations were conductedin TCNB assay buffer: 50 mM Tris, 10 mM CaCl₂), 150 mM NaCl, 0.05% (w/v)polyoxyethylene (23) lauryl ether, for example Brij-35, pH 7.5. MMPswere activated by pre-incubation, prior to use in the main assay. Toactivate rhMMP-3, the enzyme was diluted to 20 ug/mL in TCNB containing5 ug/mL chymotrypsin and incubated at 37 degrees C. for 30 minutes.Following activation, chymotrypsin activity was inhibited by theaddition of PMSF to a final concentration of 2 mM.

PMSF should not affect rhMMP-3 activity adversely. rhMMP-12 wasactivated by diluting to 50 μg/mL in TCNB and incubating at 37 degreesC. for 30 hours. No further additives or inhibitors were used toactivate rhMMP-12.

Materials

Monomer ID34F, bihead ID25F, etanercept, adalimumab and infliximab.

Methods

Test compounds, prepared in TCNB buffer, were mixed with activatedrhMMP-3 and rhMMP-12 on ice. Test compounds were present at thebeginning of the assay at a concentration of 30 ug/mL. rhMMP-3 waspresent at a starting assay concentration of 12 ug/mL. rh-MMP-12 waspresent at a starting assay concentration of 30 ug/mL. Each compound wastested in the presence of both enzymes and a TCNB buffer only control tocheck for enzyme-independent degradation over the course of the assay.Enzymes were also incubated with TCNB only to provide ‘no compound’controls. The final volume of all reactions at t=0 hours=30 uL. At t=0hours, 10 uL of the starting assay volume was removed and added to 10 uL2× protease inhibitor solution in protease stop buffer (1×PBS 2% BSA, 5mM EDTA, as described in Example 5) on ice to stop the reaction. T=0samples were frozen at −80 degrees C. The remaining 20 uL reactionvolumes were sealed in a PCR assay plate and incubated at 37 degrees C.for 22 hours.

Following 22 hour incubation the majority of the reactions were found tobe at ˜20 uL. 20 uL 2× protease inhibitor solution in protease stopbuffer (1×PBS 2% BSA, 5 mM EDTA) was added to stop the reaction. T=22 hrsamples were frozen at −80 degrees C. Assay samples were analysed byWestern blotting. Samples were diluted to the equivalent of 6.6 ng/uLtest compound in load dye and 15 uL was loaded (100 ng of each testcompound/lane, assuming no change in test compound concentration overthe course of the experiment) into a 10% Bis-Tris gel. The equivalentvolume of the ‘no test compound’ controls was also loaded. Achemiluminescent MW protein Ladder, for example Super signal (Pierce)was added as a standard at 5 uL/lane. Samples were electrophoresed inSDS-MES buffer and transferred to nitrocellulose membranes using a7minute transfer program on semi-dry blotting machine, for example, theiBlot. The membranes were blocked overnight at 4 degrees C. in blocksolution (1% BSA, 2% skim milk powder, 0.05% PEG20, 1×PBS pH7.4).Variable domains were detected using 1) Primary: Rabbit polyclonalanti-Q65B1(-Flag-6His) (terminal bleed serum) at 1/1000 in blocksolution and 2) Secondary: HRP-conjugated polyclonal Swine-anti-RabbitIgG antibody at 1/1000 plus 1% normal goat serum) in block solution.Etanercept, Adalimumab and Infliximab were detected usingperoxidase-conjugated anti-human IgG specific for Gamma-chains (Dako,P0214) at 1/1000 in block solution (no secondary antibody used). Blotswere washed for 6×5 minutes in 25 mL PBST (1×PBS, 0.1% PEG20) betweeneach incubation step to remove non-specifically bound antibody. PierceSuper-Signal ECL (34087) was used to develop the blots, which werevisualised using an ImageQuant (LAS4000), varying the exposure time,where necessary.

Results

ID34F (Q65B1 K59H and K101H variant) and ID25F (a homobihead of ID34F)appear fully resistant to both rh-MMP3 and rh-MMP12 in vitro after 22hours incubation (FIG. 5A). By comparison, each of the clinicalbiological agents Etanercept, Adalimumab, and Infliximab undergo eitherpartial, or total, cleavage of the full-length molecule to generatelower MW fragments (FIGS. 5B and 5C). The analyses show bandscorresponding to the monomer (15 kDa) and bihead (30 kDa) that areunchanged following treatment.

7.2 Evidence of Stability in Intestinal Fluids and Faecal Extracts fromthe Pig and Monkey

ID32F, ID34F, ID8F-EV and Q65B1 were each incubated in the presence ofsupernatants from (a) swine duodenum for 5 and a half hours, (b) mousesmall intestine for 5 and a half hours and (c) human faeces for 21hours. The variable domains tested showed good stability in theseextracts from different GI regions. The corresponding approximate %survival for each variable domain were as follows:

TABLE 6 Swine Mouse small Human duodenum intestine faeces ID32F 80 55 40ID34F 90 70 70 ID8F-EV 120 80 60 Q65B1 90 60 60

7.3 Stability of ID8F-EV in Extracts Prepared from Luminal Contents fromDifferent Regions of the Monkey Gastrointestinal Tract

ID8F-EV was incubated for 5 hours in stomach, duodenum, jejenum andileum supernatants, and for 16 hours in caecum and colon supernatants.After incubation, the % survival of ID8F-EV was between approximately60% and 90%.

Example 8: Local Delivery of Immunoglobulin Chain Variable Domains tothe Intestinal Tract and Access to Lamina Propria Following OralAdministration

The variable domains of the invention are unlikely to bind to murineTNF-alpha, however, demonstration of local delivery to the intestinaltract and penetration of the lamina propria following oraladministration in a mouse model of IBD provides evidence thatneutralisation of TNF-alpha may be achieved at sites of intestinalinflammation.

8.1 Colonic Epithelial Penetration of 65B1 after Ex Vivo Incubation inLumens of Colon Segments Taken from Normal and DSS Colitis Mice

Methods

DSS colitis was induced in two mice using a standard protocol. 2%dextran suphate (MP biomedical) was administered in drinking water for 7days, after which mice were kept for a further 3 days to allow peakdevelopment of disease. The mice were then killed, along with 2 normalmice, and the colons removed. Colon luminal contents were gently washedout with PBS, then segments were ligated with thread and processed asshown below. Colon segments were loaded with 3 ug/ml 65B1 in RPMIcontaining 2% FCS and 15 mM HEPES, then the open segment ends wereligated, and the segments incubated by gently rocking in RPMI+2% FCS+15mM HEPES in a culture flask at RT for 1.75h. Colon segments were thencut, washed briefly, and either fixed in paraformaldehyde, or embeddedin optimal cutting temperature compound (OCT) and snap frozen. Tissuesections were collected from the proximal colon tissue. 6 um sectionswere cut and were fixed in ice-cold acetone for 90 seconds. The sectionswere air dried and stored at −20 degrees C. until assayed. Two serialsections for each mouse were used to stain for each antibody set.

Immunohistochemistry

Slides were thawed and sections were blocked with 3% fatty acid free-BSAin PBS for 30 minutes at room temperature. Primary antibody incubation(either rabbit polyclonal anti variable domain, or a rabbit controlpolyclonal antibody) was carried out overnight (˜18 hours) at 4 degreesC. in a humidified chamber. A set of three slides for each colon tissueblock was stained as follows (one slide per treatment):

-   -   Vehicle (3% FAF-BSA in PBS as described above)    -   10 ug/ml affinity purified rabbit polyclonal control antibody    -   10 ug/ml anti-variable domain affinity purified rabbit        polyclonal antibody (AB1219).

After incubation each section was washed three times for three minuteswith ice cold-PBS. All sections were stained with a goat ant-rabbit IgGAlexa Fluor 594 antibody at 20 ug/ml (Molecular Probes A11037) and 1ug/ml Hoescht 33342 (to identify cell nuclei) in vehicle for six hoursin the dark at room temperature. Following incubation with secondaryantibody, sections were washed as described previously followed by afinal wash with Milli 0 water. Sections were air dried in the dark,mounted with an antifadant media (Citifluor, AF1) and covered with aglass coverslip. Slides were kept in the dark at 4 degrees C. untilviewed. Slides were viewed next day using an Olympus AX70 microscope andimages were captured sequentially for each flurochrome (Alexa Fluor 488and UV) using Image Pro-Plus (v7.0, Media Cybernetics). Exposure levelswere set using sections from control or DSS mice treated with thecontrol rabbit antibody polyclonal and at least two random fields ofview were captured from each slide from each animal.

Results

In contrast to the images of normal mouse colon, 65B1 associatedfluorescence is greatly increased in colon sections from DSS colitismice. The hematoxylin and eosin (H&E) section revealed extensiveinflammation, which presumably severely compromised the epithelialbarrier function, allowing ready access of 65B1 to the underlying laminapropria. Whilst there was little or no 65B1 transepithelial penetrationin colon segments from normal mice, extensive penetration occurred inthe DSS colitis mouse colon segments. The findings suggest that diseaseinduced alterations in epithelial barrier function have allowed thevariable domains access to the submucosal tissue.

8.2 Examination of Penetration of 65B1 into the Colonic Mucosa of Normaland DSS Colitis Mice after Oral Gavage

Materials and Methods

-   -   Q65B1 (3.35 mg/ml=222.3 uM)    -   1M sodium bicarbonate solution    -   Dried milk (Marvel),    -   Rabbit anti variable domain antibody, pAB 1219    -   Control rabbit pAb    -   Goat anti rabbit Alexa 594 nm (Molecular Probes, A11037)

DSS colitis was induced in 3 C57BL/6 mice by administering 2% dextransuphate (MP biomedical) in drinking water for 7 days, after which micewere kept for a further 3 days to allow peak development of disease. Onthe day of dosing, all the DSS colitis mice, and 3 normal mice weregiven 100 ul of 0.1M NaHCO₃, 450 mg/ml dried milk, then −10 minuteslater two normal mice and two DSS colitis mice with the most severedisease (as judged by body 40 weight measurements) were given 150 ul of0.1M NaHCO₃ containing 65B1, 33.6 uM final concentration (equivalent to76 ug) and 450 mg/ml dried milk, whilst the other two mice (one normaland one DSS colitis) received vehicle only. Mice were killed at3.25-3.5h post 65B1 dosing, and the gastrointestinal tracts wereremoved. Colons were isolated, the luminal contents gently squeezed out,then washed briefly in RPMI media containing 2% fetal calf serum, afterwhich they were snap-frozen as described in the enclosed protocol.

Results

Colons from mice that received no 65B1 provided material to establishbackground fluorescence against which 65B1 specific fluorescence couldbe judged. VHH associated fluorescence was red whilst the cell nuclei,labelled using Hoechst 33342, were blue. Mouse 1, which received onlyvehicle, showed the colon background fluorescence, whilst mice 2 and 3were dosed with 65B1. There is little if any difference in redfluorescence between the colons from the three mice, showing that 65B1penetration of normal mouse colon is negligible, as expected. The H&Estained section showed a typical colon structure with no obviousinflammation. In contrast to the normal mice, there were areas ofextensive inflammation and in some places significant epithelial damagehad occurred. There was a clear increase in 65B1 associated fluorescencein the DSS colitis mice receiving the variable domain compared with theDSS colitis mouse receiving vehicle only, indicating transepithelialpenetration into the lamina propria.

This result demonstrates that an orally given variable domain can accessthe colonic submucosa of mice with induced colitis. Access of thiscompartment in patients with IBD is a necessary prerequisite fortherapeutic efficacy.

Example 9: Effects of 1038F and Infliximab on the Phosphorylation ofSignalling Proteins Present in Ex Vivo Cultures of IBD Biopsy Tissue

Due to antibody-based anti-TNF-alpha therapeutics generally lackingcross-reactivity with, for example, murine TNF-alpha, it has not beenpossible to assess the efficacy of the immunoglobulin chain variabledomains of the present invention in a mouse-based preclinical model ofIBD. However, it has been shown that local intestinal delivery ofanti-murine TNF-alpha antibody domain-secreting lactobacilli issufficient to suppress colonic inflammation in a mouse model of IBD(Vandenbroucke et al 2010 Mucosal Immunology 3(1):49-56, hereinincorporated by reference in its entirety). Results of these studiesprovide preclinical validation for the concept of an orally-administeredanti-human TNF domain antibody-based approach for the prevention ortreatment of IBD.

Infliximab are effective for the treatment of IBD. Infliximab is thoughtto act primarily by neutralising the biological activity of TNF leadingto inhibition of the downstream pro-inflammatory effects of thecytokine. Activation of the many different cell types present indiseased tissue by TNF and secondary inflammatory mediators involvesmultiple receptor signalling pathways resulting in the phosphorylationof receptors, protein kinases and transcription factors. Experimentshave shown that (i) patterns of protein phosphorylation are altered inIBD vs normal intestinal tissue and that (ii) patterns ofphosphorylation are sensitive to inhibitors of specific pro-inflammatorymechanisms.

It was investigated (i) whether the TNF-neutralising activity of ID38Fcan be demonstrated in ex vivo cultures of IBD tissue based on changesin the patterns of tissue protein phosphorylation and (ii) to compareeffects of ID38F with the clinically effective anti-TNF mAb infliximab.

Organ Culture

Perendoscopic colonic biopsies or surgical ileal mucosal specimens frompatients with active IBD were placed (one biopsy per well) in 24-wellplates (VWR International, Lutterworth, UK) in 300 ul serum-free HL-1medium (Cambrex BioScience, Wokingham, UK) supplemented with 100 μ/mlpenicillin and 100 ug/ml streptomycin, and cultured at 37 degrees C.,5%002 with the following stimuli:

-   -   IgG1 10 ug/ml    -   Infliximab 10 ug/ml    -   ID38F 4 ug/ml    -   Control non-anti-TNF-alpha immunoglobulin chain variable domain)        4 ug/ml

After 48h culture, biopsies and supernatants were snap frozen and storedat −70 degrees C.

Phospho-Array Analysis Procedure

For the analysis of phospho-protein content IBD tissue samples werethawed, lysed in RIPA Buffer (Sigma-Aldrich, St. Louis, Mo.)supplemented with phosphatase inhibitor cocktail 2 (Sigma-Aldrich) andprotease inhibitor cocktail (Sigma-Aldrich), both at 1%. Proteinconcentrations of the lysates were determined by the Bio-Rad Proteinassay (Bio-Rad Laboratories, Hemel Hempstead, UK) and samples diluted to1.0 mg/ml in Array Diluent Buffer. Analysis of the phospho-proteinprofiles was performed using PathScan RTK Signalling Antibody Array Kits(Cell Signaling Technology with chemiluminescent readout #7982). Themultiplexed array is a slide-based antibody array founded upon thesandwich immunoassay principle. The array kit allows for thesimultaneous detection of 28 receptor tyrosine kinases and 11 importantsignaling nodes, when phosphorylated at tyrosine or other residues (seeTable 7A). Target-specific capture antibodies, biotinylated protein(positive control) and nonspecific IgG (negative control) were spottedin duplicate onto nitrocellulose-coated glass slides. Analysis of thetissue lysates was performed according to the manufacturer'sinstructions using the reagents provided. Briefly, each diluted lysatewas incubated on the slide followed by a biotinylated detection antibodycocktail. Streptavidin-conjugated HRP and LumiGLO® Reagent were thenused to visualize the bound detection antibody by chemiluminescence. Animage of the slide was captured on standard chemiluminescent sensitivefilm. An image of the film was obtained by scanning on a HP LaserJet Pro100 colour scanner and the spot intensities quantified using ImageJsoftware.

TABLE 7A Target Kinases and Signalling Proteins Included on the ArrayReceptor Tyrosine Kinases Signalling Nodes EGFR/ErbB1 pan-Tyr PDGFRpan-Tyr Akt/PKB/Rac Thr308 HER2/ErbB2 pan-Tyr c-Kit/SCFR pan-TyrAkt/PKB/Rac Ser473 HER3/ErbB3 pan-Tyr FLT3/Flk2 pan-Tyr p44/42 MAPK(ERK1/2) Thr202/Tyr204 FGFR1 pan-Tyr M-CSFR/CSF-1R S6 Ribosomal Proteinpan-Tyr Ser235/236 FGFR3 pan-Tyr EphA1 pan-Tyr c-Abl pan-Tyr FGFR4pan-Tyr EphA2 pan-Tyr IRS-1 pan-Tyr InsR pan-Tyr EphA3 pan-Tyr Zap-70pan-Tyr IGF-IR pan-Tyr EphB1 pan-Tyr Src pan-Tyr TrkA/NTRK1 pan-TyrEphB3 pan-Tyr Lck pan-Tyr TrkB/NTRK2 pan-Tyr EphB4 pan-Tyr Stat1 Tyr701Met/HGFR pan-Tyr Tyro3/Dtk pan-Tyr Stat3 Tyr705 Ron/MST1R pan-Tyr Axlpan-Tyr Ret pan-Tyr Tie2/TEK pan-Tyr ALK pan-Tyr VEGFR2/KDR pan-Tyr

Data Analysis

A “background” signal was measured for each blot and this was subtractedfrom the uncorrected phospho-protein signals. It was noted that negativevalues were obtained for some “background-subtracted” phospho-proteinvalues. This was likely due to the method used to read the arrays whichinvolved exposure to X-ray film and scanning of the negative imagerather than direct measurement of light output. In cases where thesignal measured for a phosphoprotein in an ID38F (or infliximab) treatedlysate gave a negative value after background correction but thecorrected value for the corresponding control non-anti-TNF-alphaimmunoglobulin chain variable domain (or IgG) treatment was positive,the level of inhibition was scored as >50%.

Visual assessment of the array data showed that both ID38F andinfliximab produced inhibitory effects on some of the phospho-proteinsin some of the tissues. To look for effects of the anti-TNF treatmentson each of the different phospho-proteins, signals taken from the ID38Fand infliximab arrays were compared directly with those from thecorresponding control variable domain and IgG arrays and the signalratios (ID38F/control variable domain and infliximab/IgG) calculated. Toassess whether there was a consistent pattern of inhibition by theanti-TNF antibodies in tissues from the different patients, thosephospho-proteins that were inhibited by 50% in tissues from at least 3of the 4 CD patients were highlighted (see Table 7B).

TABLE 7B Phospho-proteins inhibited (>50% in 3 of 4 CD Biopsies) byID38F or infliximab Phosphoproteins Inhibited (>50% in 3 of 4 IBDBiopsies) ID38F vs control Biological Functions variable domaininfliximab vs IgG Macrophage apoptotic cell AXL AXL clearance TYRO3 — TCell Signalling/Adhesion EphA3 EphA3 Lck ZAP70 Angiogenesis VEGFR2VEGFR2 TIE2 TIE2 EphB4 — Pain/Neuronal Regulation EphB1 Cell Activation/AKT AKT Survival/Signalling ERK1/2 ERK1/2 S6 Ribosomal Protein S6Ribosomal Protein — SRC ALK PDGFR Haematopoiesis FLT3 Epithelial CellRegulation EphA1 EphA1 — EphA2

Based on these criteria, analysis of the raw array data shows that ID38Ftreatment consistently inhibited 12/39 phospho-proteins while infliximabinhibited 14/39 phospho-proteins of which 8 were shared with ID38F.Functions of these proteins are all relevant to signaling pathways andimmunological processes that are likely to be important in IBDinflammation and/or pathology. When results of the analyses using the“uncorrected” and “control-normalised” data were compared the set of 12phospho-proteins inhibited by ID38F was the same in both cases; forinfliximab, 12 phosphoproteins were identified as common to bothanalyses.

To further analyse the array data, “control-normalised” values takenfrom the ID38F and infliximab arrays were compared directly with the“control-normalised” values from the corresponding control variabledomain and IgG arrays and the ratios calculated as previously. Aconsistent pattern of inhibition by the anti-TNF antibodies was againnoted based on those phospho-proteins that were inhibited by 50% intissues from at least 3 of the 4 IBD patients (see Table 7C).

TABLE 7C Phosphoproteins identified by calculation of ratios from arraydata normalised to the average positive control signal on each arrayPhosphoproteins Inhibited (>50% in 3 of 4 CD Biopsies) D38F vs controlBiological Functions variable domain infliximab vs IgB Macrophageapoptotic AXL AXL cell clearance TYRO3 — T Cell Signalling/ EphA3 EphA3Adhesion Lck — Angiogenesis VEGFR2 VEGFR2 TIE2 TIE2 EphB4 —Pain/Neuronal Regulation EphB1 RET Cell Activation/ AKT AKTSurvival/Signalling ERK1/2 ERK1/2 S6 Ribosomal Protein S6 RibosomalProtein — SRC PDGFR Haematopoiesis FLT3 cKit Epithelial Cell RegulationEphA1 EphA1 — EphA2

ID38F treatment inhibited 12/39 phospho-proteins while infliximabinhibited 14/39 phospho-proteins of which 8 were shared with ID38F. Whenresults of the analyses using the “uncorrected” and “control-normalised”data were compared the set of 12 phospho-proteins inhibited by ID38F wasthe same in both cases; for infliximab, 12 phosphoproteins wereidentified as common to both analyses.

Conclusion

In cultures of inflamed IBD tissue taken from human patients with activedisease, treatment ex vivo with ID38F or with the clinically effectivemonoclonal antibody infliximab can inhibit the phosphorylation of a setof proteins that include receptor tyrosine kinases and targets involvedin cell signaling. Evidence that treatment with structurally differentantibodies inhibited the phosphorylation of an almost identical set of 8proteins in 3 of the 4 tissues strongly suggests that the effects ofboth antibodies are due to the neutralization of endogenous TNF-drivenprocesses. Inhibition of the set of phospho-proteins by ID38F orinfliximab was seen in tissue biopsies taken from at least 3 of the 4IBD patients. Lack of inhibition of particular phospho-proteins in oneof the IBD tissues may have reflected inter-patient differences indisease and/or differences in the cellularity of biopsies taken fromdifferent sites of inflammation.

This study provides evidence that the pattern of tissue phosphoproteinsinhibited by ID38F is almost identical to that achieved with aclinically relevant concentration of infliximab.

Example 10: Neutralising Potency Comparison of 1038F (a PolypeptideAccording to the Present Invention) and Anti-TNF-Alpha Polypeptides ofthe Prior Art

The neutralising potency of ID38F was compared in one and the same assayto that of the following anti-TNF-alpha polypeptides of the prior art:

TNF1 (a VHH disclosed in WO2006122786, therein SEQ ID NO: 52)

TNF3 (a VHH disclosed in WO2006122786, therein SEQ ID NO: 60)

TNF30 (a VHH disclosed in WO2006122786, therein SEQ ID NO: 96)

VHH#3E (a VHH disclosed in WO2004041862, therein SEQ ID NO: 4)

Adalimumab (commercially available human monoclonal antibody)

Materials

L929 cells (10⁴ cells/well)

96-well plates (Costar)

DMEM (Invitrogen) supplemented with Pen/Strep+2 mM L-glutamine

Human TNF-alpha (Invitrogen) concentration: 500 μg/ml

Actinomycin D concentration (Sigma): 0.75 ug/mL

ID38F purified from S. cerevisiae

ID38F Flag-His tagged purified from E. coli

Adalimumab

TNF1, TNF3, TNF30, VHH#4E purified from E. coli with Flag-His tags

Range of dilutions: 5 μM-30 nM (1:3 dilutions)

Incubation times: 23h

Alamar Blue cell viability reagent (Invitrogen, DAL1100): 10 uL/well 3%SDS

Microplate reader (Fluostar Optima) (OD590 nm)

Method

10⁴ L929 cells/well in 100 ul were plated on day 0 in 96 wellmicro-plates (Costar) in DMEM complete medium and stored over night at37° C. and 5% CO₂. On day 1 serial dilutions 1:3 (in DMEM+Act.D) foreach purified VHH/Ab were set up at doubled the assay concentrations(with volumes sufficient for triplicates) starting from a topconcentration of 60 nM. 165 uL of each dilution were then diluted 1:1with 165 uL of hTNF-alpha 2× (1 ng/mL) prepared in DMEM+Act.D. 0.9 mL ofTNF 2× were diluted with 0.9 mL of CM+Act. D to have the TNF onlycontrol in the assay. The medium was removed from each well of the assaymicroplates and the cells were incubated with 100 uL of each TNF+VHHdilution, CM+act. D or TNF only control. After 23h of incubation at 37°C. and 5% CO₂, 10 ul of Alamar Blue were added to each well, the cellswere incubated for 2h at 37° C. and 5% CO₂, and 50 ul of 3% SDS weresubsequently added to each well. The plates were then read at 590 nm.

Results

The resultant neutralisation curves (produced with GraphPad Prism, using4 parameter non-linear regression curve) are shown in FIG. 6. EC50values are shown in Table 8.

TABLE 8 Anti-TNF-alpha polypeptide EC50 (nM) TNF1 0.751 TN F3 0.631 TNF30 0.420 VHH#3E 1.110 ID38F-Flag-His (E. coli-produced) 0.102 ID38F (S.cerevisiae-produced) 0.127 Adalimumab 0.091

It can be seen from FIG. 6 and Table 8 that VHH#3E was the least potentanti-TNF-alpha polypeptide in neutralising human solubleTNF-alpha-induced cytotoxicity in L929 cells. ID38F (both E. coli- andS. cerevisiae-produced) had an approximately 4 fold −1 log lower EC50than that of the prior art anti-TNF-alpha VHHs.

Example 11: Formulation of an Anti-TNF-Alpha ICVD of the Invention intoa Pharmaceutical Composition

A solid pharmaceutical composition comprising one of the anti-TNF-alphaICVDs of the invention disclosed herein was produced in the form ofmini-tablets by dry granulation and compression. This anti-TNF-alphaICVD is a 115 amino acid, 12.6 kDa polypeptide with a pl of 6.8 and anaqueous solubility of greater than 30 mg/mL. The ICVD binds with highaffinity to, and has potent neutralising activity against, human andCynomolgus monkey TNF-alpha.

The mini-tablets were presented in different presentations, wherein eachpresentation contained a different quantity of mini tablets in differentsizes of capsules. The main presentation used in the examples detailedbelow was a size 00 HPMC capsule containing 15 enterically coatedmini-tablets (total 185 mg of pharmaceutically active bindingpolypeptide). The mini-tablet cores had a diameter of 3 mm (excludingcoating thickness). The components contained in each mini-tablet andtherefore in the 15 mini-tablets contained in the capsule are listed inTable 9 below.

TABLE 9 Quantity (mg/capsule) Quantity (mg) Name of mini tablet % w/w in185 mg dose 12 mg dose component Function composition (15 mini-tablets)(1 mini-tablet) Mini-tablet cores Total polypeptide Active 45.7 225 15pharmaceutic al ingredient (API) Mannitol Compression 12.0 59.25 3.95aid Microcrystalline Compression 14.6 72 4.8 cellulose aidCroscarmellose Super 3.1 15 1 sodium disintegrant Magnesium stearateLubricant 0.8 3.75 0.25 Sub coating Hydroxypropylmethyl Polymer coat 3.818.75 1.25 cellulose pH sensitive enteric coating Eudragit ® L100Enteric 11.7 57.76 3.85 polymer coat Triethyl citrate Plasticiser 2.311.51 0.77 Talc Anti-tacking 5.9 28.93 1.93 agent Sodium laurylSurfactant 0.04 0.20 0.01 sulphate

The total polypeptide in the composition has a purity of approximately70-90% such that 225 mg of polypeptide contains 185 mg ofpharmaceutically active binding polypeptide.

The mini-tablets were produced by the following methodology.

The lyophilised polypeptide was blended with mannitol and a portion ofthe magnesium stearate and dry slugged to increase its density. Thismaterial was then passed through a screen, blended with the othermini-tablet excipients (microcrystalline cellulose, croscarmellosesodium and the remaining magnesium stearate) and compressed to producethe mini-tablets.

The mini-tablets were then coated with a 5% solution of hydroxylpropylmethyl cellulose in ethanol:water 80:20, dried and the solvent removedto create a sub-coat and a smoother surface. The mini-tablets were thencoated with Eudragit® L100 polymer, together with triethyl citrate, talcand sodium lauryl sulphate, as an organic solution in isopropyl alcoholand water and dried to create a pH-sensitive enteric coat. The resultingapproximately 3 mm diameter mini-tablets were then filled into capsulesat the doses given above. The pH sensitive enteric coating had athickness of 100-170 um. The anti-TNF-alpha ICVD of the invention usedin this formulation is also referred to below as “pharmaceuticallyactive binding polypeptide”, “ICVD” and “polypeptide”.

Example 12: Administration to Cynomolgus Monkeys: PolypeptideConcentration in Different Intestinal Tract Compartments and in Faeces

12.1 Polypeptide Concentration in Different Intestinal TractCompartments

A study was conducted to assess the release profile of a compositionsimilar to that of Example 11 through regions of the intestinal tractwhen orally administered to female Cynomolgus monkeys. The releaseprofile was assessed by analysis of polypeptide concentration in thedifferent intestinal tract compartments.

A single capsule containing 11 mini-tablets was administered orally toeach of three Cynomolgus monkeys (the monkeys are referred to as M234,M236 and M238). The mini-tablet composition varied from that of Example11 in that each mini-tablet contained an additional 1 mg of methyleneblue (dye) and a dose of 141 mg of the ICVD. 8 of the mini-tablets alsocontained 0.7 mg of isoprenalin. The methylene blue dye was for visualanalysis of the distribution of dissolved mini-tablets through thegastrointestinal (GI) tract (not discussed herein) and the isoprenalinwas for use in a study monitoring heart rate (not discussed herein).

Four hours after oral dosing, the animals were culled. Thegastrointestinal tracts were carefully removed, the different GIcompartments ligated then cut and the luminal contents and washescollected. The number of undissolved and partially dissolved minitablets were noted and these mini tablets were removed. The samples werethen homogenised and frozen until analysis. After initial centrifugationof the slurries for 5 min at 5000 rpm at 10° C., 1 ml of supernatant wasremoved from each sample and centrifuged at 13300 rpm in a microfuge atthe same temperature for 5 min. The supernatants were then centrifugedagain under the same conditions, but for 20 min, after which, they wereanalysed using a standard Humira competition ELISA. All dilutions ofsamples and Humira and the ICVD standard were prepared in PBS containing1% BSA, 0.6M NaCl, 1% human AB serum, 0.05% Tween 20 and 2× proteaseinhibitors. ICVD concentrations were interpolated from a standard curveusing a 4 parameter, non-linear curve fitting equation in GraphPadPrism. ICVD concentrations in undiluted GI tract samples were derived bytaking the means of the best interpolated data multiplied by thesupernatant dilution factor.

No intact mini-tablets were found in the stomach, duodenum, jejunum orileum of either M236 or M238. In M234, 4 intact mini-tablets were foundin the stomach, 1 in the duodenum and 1 in the jejunum. No partiallydissolved mini-tablets were found in any GI tract region of any monkey.

Preparation of the slurry supernatants necessitated adding large volumesof buffer, inevitably diluting the ICVD. In FIG. 7, the expected luminalconcentrations of ICVD are presented. These were calculated, assumingthat the luminal GI tract contents have a specific gravity of 1, bymultiplying the supernatant ICVD concentrations by the fold dilution onaddition of buffer. As shown, very high ICVD concentrations (0.1->1 mM)are likely to occur in the lumen of some monkey GI tract compartments.

ICVD was only detected in the contents of one Cynomolgus monkey stomach(M234). ICVD was also found at high concentrations in the contents ofthe ileum, caecum and upper colon of all monkeys. In addition, M234 andM238 were detected at high concentrations in the contents of the jejunum(see FIG. 7).

Finally, the % ICVD recovered was calculated, assuming the actual doseat 4h was delivered by only mini-tablets that had dissolved. As shown inFIG. 8, between 51.5 and 74.9% of the ICVD dose was accounted for.

This study has shown that an anti-TNF-alpha ICVD of the invention can bedelivered at high concentrations to the lower GI tract of Cynomolgusmonkeys. The finding that some mini-tablets remained intact 4h afterdosing suggests that the dose will be delivered over a period of time,offering the potential of prolonged exposure. If these findings aremirrored in treatment of IBD patients when using an anti-TNF-alphabinding polypeptide, then it is reasonable to expect that theconcentrations of anti-TNF-alpha polypeptide exposed to the lower GItract will be more than adequate for effective TNF-alpha neutralisation.

12.2 Polypeptide Concentration in Faeces

A single capsule containing 11 mini-tablets was administered orally toeach of three Cynomolgus monkeys. The mini-tablet composition variedfrom that of Example 11 in that each mini-tablet contained an additional1 mg of methylene blue (dye) and 8 of the mini-tablets also contained0.7 mg of isoprenalin. The methylene blue dye was for visual analysis ofthe dissolution of mini-tablets in faeces and the isoprenalin was foruse in a study monitoring heart rate (not discussed herein).

Pooled faeces from the monkeys were collected at 8, 12, 20, 24 and 36h(no samples were collected at 16h). No mini-tablets were found in any ofthe faecal samples. These were mixed with extraction buffer (0.1% BSA,0.6M NaCl, 0.05% Tween 20, lx protease inhibitors, 5 mM EDTA in PBS), at1 g faeces/4 ml buffer, then homogenised and the slurries frozen at −80°C. for storage before analysis. Visual examination revealed bluecolouration of the 12h, 20h, 24h and 36h slurries. Previous in vitroexperiments (not shown) have demonstrated that the increasing methyleneblue concentration upon dissolution of the mini-tablets is closelycorrelated with ICVD concentration.

Slurries were thawed and centrifuged for 5 min at 4,000 rpm (3,200 g) toremove the bulk of particulate matter. About 1 ml of each supernatantwas transferred to Eppendorf tubes and centrifuged in a microfuge at13.5K, 10° C. for 5 min, after which supernatants were placed in newtubes and centrifuged for 20 min at 10° C. Supernatants were then usedimmediately for ICVD measurement using a Humira competition ELISA.

The ELISA OD450 readings for the different faecal supernatants are shownin FIG. 9. The data clearly show that ICVD is present in the faecessupernatant samples at all time points, with the possible exception ofthe 36h supernatant (though there may be slight activity visible at thelowest dilution).

Interpolating these data against standard curves for ICVD using GraphPadPrism and multiplication by the dilution factor of buffer added gave theICVD concentrations in each faecal sample, using the assumptions that 1g faeces is equivalent to 1 mL liquid volume and that the polypeptide isuniformly distributed in the faeces. These are shown in FIG. 10.

Using slurry volumes (calculated on the basis of 1 g faeces=1 ml,+volume of buffer for extraction) the pg amounts of ICVD in each samplewere determined (FIG. 11)).

In summary, a sustained substantial concentration of pharmaceuticallyactive binding polypeptide was achieved through the cynomolgus monkeyintestinal tract for greater than 8 hours.

Example 13: Administration to Humans: Polypeptide Concentration at theIleal-Caecal Junction and in Faeces

13.1 Polypeptide Concentration at the Ileal-Caecal Junction

The aim of this study was to demonstrate that the anti-TNF-alpha ICVD ofthe invention incorporated into the composition of Example 11 isdelivered at high concentrations to the ileal-caecal junction in man, amajor site for Crohn's disease and the proximal site of Crohn's diseaselesions in the intestine of many patients.

Four human volunteers, fitted with terminal ileostomy bags each receiveda single oral dose of 1665 mg ICVD, formulated into mini-tabs insidesize 00 capsules (9 capsules in total). In these otherwise healthyindividuals, the entire contents of the terminal ileum drains into thedetachable external bag. At each hourly time point post-dosing, thefitted bag containing the total ileal effluent was removed, frozen and anew bag was fitted. Ileostomy samples were collected in this mannerevery hour for a period of 12 hours post dosing. Following this time,ileostomy samples were collected every four hours up to 24 hours postdosing. A Pre-dosing sample (day −1) was also taken as a control. Anypartially dissolved mini-tablets observed in the bags were removed priorto analysis such that only fully soluble ICVD was analysed. The ICVD wasextracted from the ileal fluid and concentrations of active ICVD weredetermined by functional ELISA, assuming that 1 g ileal fluid isequivalent to 1 mL liquid volume.

The data revealed high concentrations of active ICVD present in theileostomy bags, in the range 200 nM up to 1 mM. In addition, highconcentrations were observed over several hours of bag changes for eachsubject (see Table 10).

TABLE 10 Hour ICVD concentration in Subject post dose ileal fluid (nM)31001 2 406350 31001 3 305560 31001 4 791 31002 2 32780 31002 3 113000031002 4 792060 31002 5 81750 31002 6 12780 31002 7 1300 31002 8 42231002 9 1410 31002 10 7520 31002 11 10080 31002 12 9210 31002 16 698031003 3 1060000 31003 4 496030 31003 5 7080 31003 8 46110 31003 9 7548031003 10 16030 31003 11 72940 31003 12 15870 31003 16 881 31004 2 12619031004 3 235 31004 4 11110 31004 5 3770 31004 6 6730 ICVD was notdetected in any of the predose (Day −1) samples from any subject.

In summary, a sustained and high concentration of pharmaceuticallyactive binding polypeptide was achieved at the ileal-caecal junction inthese human volunteers.

13.2 Polypeptide Concentration in Faeces

Healthy male subjects aged 18-45 were dosed orally with a single dose ofeither 62, 555, 1665 or 4995 mg of ICVD, using the composition detailedin Example 11. Each single dose per subject was administered between8:30 to 12:00 on day 1. Faecal samples were collected pre dose (eitheron day −1, or prior to dosing on day 1) and at all available times postdosing up to the morning of day 4 (the end of the study). ICVD wasextracted from the faeces and concentrations of active ICVD weredetermined by functional ELISA, assuming that 1 g faeces is equivalentto 1 mL liquid volume and that the polypeptide is uniformly distributedin the faeces.

High concentrations in the range 180 nM to 724 μM were obtained in thefaeces of subjects (see Table 11).

TABLE 11 Faecal mg sample Subject dose collection Pre or post [ICVD] inID ICVD day dose faeces (nM) 11001 62 −1 PRE DOSE 0 11001 1 POST DOSE1013 13001 555 −1 PRE DOSE 0 13001 2 POST DOSE 1052 13003 555 −1 PREDOSE 0 13003 1 POST DOSE 1938 13003 2 POST DOSE 1511 14002 1665 −1 PREDOSE 0 14002 1 POST DOSE 5491 14002 2 POST DOSE 558 14004 1665 −1 PREDOSE 0 14004 2 POST DOSE 27532 14006 1665 −1 PRE DOSE 0 14006 2 POSTDOSE 62579 15001 4995 −1 PREDOSE 0 15001 1 POST DOSE 10047 15001 2 POSTDOSE 135285 15001 3 POST DOSE 330 15004 4995 −1 PREDOSE 0 15004 3 POSTDOSE 273 15005 4995 1 PRE DOSE 0 15005 1 POST DOSE 724684 15005 2 POSTDOSE 258703 15005 3 POST DOSE 3536 15006 4995 −1 PRE DOSE 0 15006 1 POSTDOSE 57120 15006 2 POST DOSE 358 15006 2 POST DOSE 186

Anti-TNF agents that are used clinically to treat Crohn's disease, suchas adalimumab (Humira) and infliximab (Remicade), are administeredeither by intravenous infusion or subcutaneous injection. Ungar et al.(2016) Clin Gastroenterol Hepatol. 14(4):550-557 state that trough serumlevels of 56-83 nM (8-12 μg/mL) for adalimumab and 42-70 nM (6-10 μg/mL)for infliximab are required to achieve mucosal healing in 80%-90% ofpatients with IBD, and that this could be considered as a “therapeuticwindow”. These trough serum levels are also indicated in FIG. 7 inrespect of calculated luminal anti-TN F-alpha ICVD concentrations incynomolgus monkey gastrointestinal tract sections established aboveunder point 12.1.

Concentrations of anti-TNF-alpha ICVD delivered to the ileal-caecaljunction (section 13.1 above) and recovered in the faeces of humanvolunteers during the clinical work in this section were significantlyhigher than these levels and are thus predicted to be efficacious as atreatment for Crohn's disease. This assumes that gut luminalconcentrations of anti-TNF-alpha ICVD are comparable to serumconcentrations of marketed anti-TN F agents with respect toaccess/penetration to the gut mucosa and sub-mucosa. However, it hasbeen demonstrated in further experimental work (not shown) that thisanti-TNF-alpha ICVD of the invention, dosed orally in DSS colitis mice,is able to penetrate to the lamina propia where it is resident forseveral hours, despite a lack of target (TNF) engagement in mice.

Taken together with the data presented under 13.1 above, these resultsdemonstrate successful delivery of therapeutic levels of ICVD from theileal-caecal junction to the anus.

Example 14: Administration to Humans: Immunogenicity Study

Protein drugs, including therapeutic antibodies, may elicit an antibodyresponse in patients. Antibodies (of multiple Ig classes) produced inpatients that recognise epitopes of protein drugs are termed anti-drugantibodies (ADAs). The presence of ADAs can result in loss of drugefficacy/potency or adverse patient effects (van Schie et al., Ann RheumDis 2015 74:311-314).

A study was undertaken to assess whether sustained oral dosing in man ofthe composition of Example 11 elicits an ADA response. Healthy malesubjects aged 18-45 were dosed orally, three times daily, for 14 dayswith capsules containing 1665 mg (a total of 4995 mg per day) ICVD orplacebo, formulated into mini-tabs according to Example 11. Serumsamples from subjects were taken prior to dosing, at days 7 and 14post-dosing, and finally at 28 days (14 days after treatment cessation).These samples were analysed by Sandwich ELISA for the presence of ICVDanti-drug antibodies (ADA). This analysis revealed ADA positive sera,albeit at low titres, from 4 volunteers, two of whom received placebo.In all of these individuals ADAs were present at some level prior toICVD dosing (pre-existing ADAs).

Analysis of ICVD potency in a TNF-TNFR2 ELISA revealed that the presenceof all ADA-positive human sera samples at 5% did not affect ICVDactivity against TNF-alpha. Therefore, no evidence of ICVD neutralisingADAs was found in the sera of any volunteer at any timepoint (see Table12).

TABLE 12 ADA sandwich ADA ICVD Subject Active or ELISA Titre/serumneutral- ID placebo Sample screening dilution isation 21001 ActivePredose Negative 21001 Active Day 7 Negative 21001 Active Day 14Negative 21001 Active Day 28 Negative 21002 Active Predose Negative21002 Active Day 7 Negative 21002 Active Day 14 Negative 21002 ActiveDay 28 Negative 21003 Active Predose Negative 21003 Active Day 7Negative 21003 Active Day 14 Negative 21003 Active Day 28 Negative 21004Placebo Predose Positive 64 No 21004 Placebo Day 7 Positive 64 No 21004Placebo Day 14 Positive 64 No 21004 Placebo Day 28 Positive 64 No 21005Active Predose Positive 64 No 21005 Active Day 7 Positive 32 No 21005Active Day 14 Positive 32 No 21005 Active Day 28 Positive 32 No 21006Active Predose Negative 21006 Active Day 7 Negative 21006 Active Day 14Negative 21006 Active Day 28 Negative 21007 Active Predose Negative21007 Active Day 7 Negative 21007 Active Day 14 Negative 21007 ActiveDay 28 Negative 21008 Active Predose Positive 4 No 21008 Active Day 7Positive 4 No 21008 Active Day 14 Positive 8 No 21008 Active Day 28Positive 128 No 21009 Placebo Predose Positive 8 No 21009 Placebo Day 7Positive 8 No 21009 Placebo Day 14 Positive 16 No 21009 Placebo Day 28Positive 8 No 21010 Active Predose Negative 21010 Active Day 7 Negative21010 Active Day 14 Negative 21010 Active Day 28 Negative

CLAUSES

A set of clauses defining the invention and its preferred aspects is asfollows:

-   1. A polypeptide comprising an immunoglobulin chain variable domain    which binds to TNF-alpha, wherein the immunoglobulin chain variable    domain comprises three complementarity determining regions    (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1    comprises a sequence sharing 60% or greater sequence identity with    SEQ ID NO: 1, CDR2 comprises a sequence sharing 50% or greater    sequence identity with SEQ ID NO: 2 and    -   (a) CDR3 comprises a sequence sharing 80% or greater sequence        identity with SEQ ID NO: 3 or    -   (b) CDR3 comprises a sequence sharing 50% or greater sequence        identity with SEQ ID NO: 3 and wherein the residue of CDR3        corresponding to residue number 3 of SEQ ID NO: 3 is R, D, N, C,        E, Q, G, H, I, L, K, M, F, P, S, T, W, Y or V.-   2. The polypeptide according to clause 1, wherein the residue of    CDR3 corresponding to residue number 3 of SEQ ID NO: 3 is R, H, D,    E, N, Q, S, T, Y, G, V, L, W, P, M, C, F or-   3. The polypeptide according to clause 2, wherein the residue of    CDR3 corresponding to residue number 3 of SEQ ID NO: 3 is H.-   4. The polypeptide according to clause 3, wherein CDR3 comprises SEQ    ID NO: 3.-   5. The polypeptide according to clause 1, wherein the sequence of    CDR3 is SEQ ID NO: 3, SEQ ID NO: 70, SEQ ID NO: 71 or SEQ ID NO: 72.-   6. The polypeptide according to any one of clauses 1 to 5, wherein    CDR1 comprises a sequence sharing 80% or greater sequence identity    with SEQ ID NO: 1.-   7. The polypeptide according to clause 6, wherein CDR1 comprises SEQ    ID NO: 1.-   8. The polypeptide according to clause 6, wherein the sequence of    CDR1 is SEQ ID NO: 1, SEQ ID NO: 59 or SEQ ID NO: 60.-   9. The polypeptide according to any one of clauses 1 to 8, wherein    CDR2 comprises sequence sharing 55% or greater sequence identity,    such as sharing 60% or greater sequence identity, such as sharing    70% or greater sequence identity, such as sharing 75% or greater    sequence identity, such as sharing 870% or greater sequence    identity, such as sharing 85% or greater sequence identity, such as    sharing 90% or greater sequence identity, with SEQ ID NO: 2.-   10. The polypeptide according to clause 9, wherein CDR2 comprises    SEQ ID NO: 2.-   11. The polypeptide according to clause 9, wherein the sequence of    CDR2 is SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 2, SEQ ID NO: 63,    SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID    NO: 68, SEQ ID NO: 69.-   12. The polypeptide according to any one of clauses 1 to 11, which    comprises a sequence sharing 50% or greater sequence identity, such    as sharing 55% or greater sequence identity, such as sharing 60% or    greater sequence identity, such as sharing 65% or greater sequence    identity, such as sharing 70% or greater sequence identity, such as    sharing 75% or greater sequence identity, such as sharing 870% or    greater sequence identity, such as sharing 85% or greater sequence    identity, such as sharing 90% or greater sequence identity, such as    sharing 95% or greater sequence identity, such as sharing 96% or    greater sequence identity, such as sharing 97% or greater sequence    identity, such as sharing 98% or greater sequence identity, such as    sharing 99% or greater sequence identity, with SEQ ID NO: 8.-   13. The polypeptide according to clause 12 which comprises SEQ ID    NO: 8.-   14. The polypeptide according to any one of clauses 1 to 13, which    is selected from the list consisting of: a VHH, a VH, a VL, A V-NAR,    a Fab fragment and a F(ab′)2 fragment.-   15. A polynucleotide encoding the polypeptide according to any of    clauses 1 to 14, especially wherein the polynucleotide comprises or    consists of a sequence sharing 70% or greater, such as 80% or    greater, such as 90% or greater, such as 95% or greater, such as 99%    or greater sequence identity with any one of SEQ ID NOs: 83 to 88,    or more especially wherein the polynucleotide comprises or consists    of any of of SEQ ID NOs: 83 to 88.

MISCELLANEOUS

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the following claims

SEQUENCES Name (SEQ ID NO) FR1 CDR1 FR2 CDR2 Family 1 sequences Q65F2EVQLVESGGGLVQPGGSLRLSCAASGFDFS SHWMY WVRQAPGKGLEWVS EINTNGLITSYVDSVKG(23) Q65F3 EVQLVESGGGLVQPGGSLRLSCAASGFDFS SHWMY WVRQAPGKGLEWVSEINTNGLITKYIDSVRG (24) Q62F2 QVQLVESGGGLVQPGGSLRLSCAASGFDFN SHWMYWVRQAPGKGLEWVS EINTNGLITNYVDSVKG (25) Q65G1EVQLVESGGGLVQPGGSLRLSCVASGFDFY SHWMY WVRQAPGKGLEWVS EINTNGLITKYIDSVRG(26) Q65H6 EVQLVESGGGLVQPGGSLRLSCVASGFDFY SHWMY WVRQAPGKGLEWVSEINTNGLITKYIDSVRG (27) Q65F1 EVQLVESGGGLVQPGGSLRLSCAASGFDFG VHWMYWVRQAPGKGLEWVS EINTNGLITKYIDSVGG (28) Q65D1EVQLVESGGGLVQPGGSLRLSCTASGFDFD NHWMC WVRQAPGKGLEWVS EINTNGLITKYADFVKG(29) Q65C7 EVQLVESGGGLVQPGRSLRLSCTASGFDFS SHWMY WVRQAPGKGLEWVSEINTNGLITKYADFVKG (30) Q65D3 EVQLVESGGGLVQPGGSLRLSCVASGFDFS SHWMYWVRQAPGKGLEWVS EINTNGLITKYADSTKG (31) Q65B1EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKG(32) Family 2 sequences Q65F6 EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMYWVRQAPGKELEWVA EINTNGLITLYSDSVRG (33) Q65F11EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMY WVRQAPGKELEWVA EINTNGLITLYADSVKG(34) Q65E12 EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMY WVRQAPGKELEWVAEINTNGLITLYADSVKG (35) Q65C12 EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMYWVRQAPGKELEWVA EINTNALITTYADSVKG (36) Q65A6EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMY WVRQAPGKELEWVA EINTNGLITHYTDSVSG(37) Q65A3 EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMY WFRQAPGKELEWVAEINTNALITKYADSVKG (38) Q62E10 EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMYWFRQAPGKELEWVA EINTNALITKYADSVKG (22) Q62F10EVQLVESGGGLVQPGGSLRLSCTASGFDFG IHWMY WVRQAPGKELEWVA EINTNGLITVYPDSVKG(39) Q65B1-based constructs and mutants ID7F-EVEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKG(41) ID9F-EV EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITKYGDSVHG (43) ID13F-EV EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMYWVRQAPGKELEWLS EINTNGLITKYGDSVKG (44) ID14F-EVEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKG(45) ID15F-EV EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITKYGDSVKG (46) ID22F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMYWVRQAPGKELEWLS EINTNGLITKYGDSVKG (47) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVKGID23F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVKG (48) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVKGID24F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVKG (49) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVKGID25F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVKG (50) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVKGID26F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVKG (51) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVKGID27F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVHG (52) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVHGID28F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVHG (53) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVHGID29F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITKYGDSVHG (54) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITKYGDSVHGID8F-EV EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITKYGDSVKG (42) ID34F EVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMYWVRQAPGKELEWLS EINTNGLITHYGDSVKG (56) ID37FDVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLITHYGDSVKG(57) ID38F DVQLVESGGGLVQPGASLKLSCAASGFDFS SHWMY WVRQAPGKELEWLSEINTNGLITHYGDSVKG  (8) Comparative examples Q62F11QVQLVESGGGLVQPGGSLRLSCAASGFSFS DYVMG WFRQAPGKEREFVG FIRWDGLVTRYADAVKG(40) Name (SEQ ID NO) FR3 CDR3 FR4 Family 1 sequences Q65F2RFTVSRDNAANTLYLEMTSLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (23) Q65F3RFTASRDNAANTLYLEMTNLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (24) Q62F2RFTVSRDNAANTLYLEMTSLKPEDTALYYCAR NQKGLN KGQGTQVTVSS (25) Q65G1RFTVSRDNAANTLYLEMTNLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (26) Q65H6RFTVSRDNAANTLYLEMTNLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (27) Q65F1RFTVSRDNAANRLYLEMTNLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (28) Q65D1RFTVSRDNAANTLYLQITRLKPEDTALYYCAR NQKGLN KGQGTQVTVSS (29) Q65C7RFTVSRDNAANTLYLEITRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (30) Q65D3RFTVSRDNAANMLNLEMTSLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (31) Q65B1RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (32)Family 2 sequences Q65F6 RFTASRDNANNALFLQMNDLKFEDTAVYYCAK SRNGAAKGQGTQVTVSS (33) Q65F11 RFTASRDNANNALFLQMNDLKFEDTAVYYCAK ARNGAAKGQGTQVTVSS (34) Q65E12 RFTASRDNANNALFLQMNDLKFEDTAVYYCAK SRNGAAGGQGTQVTVSS (35) Q65C12 RFTISRDNANNTLFLQMNDLKFEDTAVYYCTH SRNGAAKGQGTQVTVSS (36) Q65A6 RFTISRDNANNTLFLQMNDLKFEDTAVYACAT SRNGAAKGQGTQVTVSS (37) Q65A3 RFTISRDNANNTLFLQMNDLKSEDTAVYYCSN TQNGAAKGQGTQVTVSS (38) Q62E10 RFTISRDNANNTLFLQMNDLKSEDTAVYYCSN TQNGAAKGQGTQVTVSS (22) Q62F10 RFTISRDNANNTLFLQMNNLKFEDTAVYYCTN TQNGKTKGQGTQVTVSS (39) Q65B1-based constructs and mutants ID7F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (41) ID9F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (43) ID13F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS (44) ID14F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN HGQGTQVTVSS (45) ID15F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN HGQGTQVTVSS (46) ID22FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (47)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID23FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (48)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID24FRFTVSRNNAANSMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (49)RFTVSRNNAANSMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID25FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS (50)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS ID26FRFTVSRNNAANSMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS (51)RFTVSRNNAANSMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS ID27FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (52)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID28FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (53)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS ID29FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS (54)RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS ID8F-EVRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (42) ID34FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS (56) ID37FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (57) ID38FRFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQHGLN KGQGTQVTVSS  (8)Comparative examples Q62F11 RFTISRDNARNTLSLQTIGLLAEDTAVYYCAASGGGSGPVNAGSYEY WGQGTQVTVSS (40)

1. A construct comprising at least one polypeptide which is animmunoglobulin chain variable domain which binds to TNF-alpha andcomprises a set of three complementarity determining regions (CDRs)comprising CDR1, CDR2 and CDR3, wherein the respective SEQ ID Nos ofCDR1, CDR2 and CDR3 of said set are selected from the group consistingof: (a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, (b) SEQ ID NO: 1,SEQ ID NO: 63 and SEQ ID NO: 70, (c) SEQ ID NO: 1, SEQ ID NO: 64 and SEQID NO: 70, (d) SEQ ID NO: 1, SEQ ID NO: 65 and SEQ ID NO: 70, (e) SEQ IDNO: 59, SEQ ID NO: 66 and SEQ ID NO: 71, (f) SEQ ID NO: 60, SEQ ID NO:67, and SEQ ID NO: 70, (g) SEQ ID NO: 1, SEQ ID NO: 67 and SEQ ID NO:70, (h) SEQ ID NO: 1, SEQ ID NO: 68 and SEQ ID NO: 72, (i) SEQ ID NO: 1,SEQ ID NO: 69 and SEQ ID NO: 70, (j) SEQ ID NO: 1, SEQ ID NO: 62 and SEQID NO: 70, (k) SEQ ID NO: 1, SEQ ID NO: 69 and SEQ ID NO: 3, (l) SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 70, (m) SEQ ID NO: 1, SEQ ID NO: 61and SEQ ID NO: 70 and (n) SEQ ID NO: 1, SEQ ID NO: 62 and SEQ ID NO: 3;and at least one different polypeptide.
 2. The construct according toclaim 1 wherein the immunoglobulin chain variable domain is a heavychain variable domain.
 3. The construct according to claim 2 wherein theheavy chain variable domain comprises a sequence sharing 90% or greatersequence identity with SEQ ID NO:
 8. 4. The construct according to claim3 wherein the heavy chain variable domain comprises a sequence sharing99% or greater sequence identity with SEQ ID NO:
 8. 5. The constructaccording to claim 4 wherein the heavy chain variable domain comprisesSEQ ID NO:
 8. 6. The construct according to claim 2 wherein the heavychain variable domain consists of a sequence sharing 90% or greatersequence identity with SEQ ID NO:
 8. 7. The construct according to claim3 wherein the heavy chain variable domain consists of a sequence sharing99% or greater sequence identity with SEQ ID NO:
 8. 8. The constructaccording to claim 7 wherein the heavy chain variable domain consists ofSEQ ID NO:
 8. 9. The construct according to claim 1 wherein thedifferent polypeptide is an immunoglobulin chain variable domain. 10.The construct according to claim 9 wherein the different polypeptidewhich is an immunoglobulin chain variable domain is a heavy chainvariable domain.
 11. The construct according to claim 1 wherein thedifferent polypeptide binds to IL-7R.
 12. The construct according toclaim 1 wherein the different polypeptide binds to IL-23.
 13. Theconstruct according to claim 1 wherein the polypeptides are connected byat least one linker.
 14. The construct according to claim 13 wherein thepolypeptides are connected by at least one protease-labile linker. 15.The construct according to claim 13 wherein the polypeptides are allconnected by non-protease-labile linkers.
 16. The construct according toclaim 1 which is substantially resistant to one or more proteasespresent in the stomach or the small or large intestine.
 17. Theconstruct according to claim 1 comprised within a pharmaceuticalcomposition with one or more pharmaceutically acceptable diluents orcarriers.
 18. The construct according to claim 1 which neutralises humanTNF-alpha cytotoxicity in the normal L929 assay with an EC50 of 0.5 nMor less.
 19. A method of treating autoimmune and/or inflammatory diseasecomprising administering to a person in need thereof a therapeuticallyeffective amount of the polypeptide of claim
 1. 20. The method oftreating autoimmune and/or inflammatory disease according to claim 19wherein the polypeptide is administered orally.